Genes encoding plant transcription factors

ABSTRACT

According to the present invention, a protein which binds to DRE to activate the transcription of genes located downstream of the DRE, a gene encoding the protein, a recombinant vector comprising the gene, a transformant comprising the recombinant vector, a transgenic plant comprising the gene, and a method for producing the gene using the transformant are provided. The present invention is useful for creating a stress tolerant plant.

This application is related to the co-pending application, ENVIRONMENTALSTRESS-TOLERANT PLANTS, U.S. application Ser. No. 09/301,217, filed onApr. 28, 1999.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a protein which binds to a stressresponsive element and regulates the transcription of genes locateddownstream of the element; a gene coding for the above protein; arecombinant vector comprising the gene; a transformant comprising therecombinant vector; a transgenic plant comprising the gene; a method forproducing the above protein using the transformant; and a method fordetermining a stress level in a plant.

2. Prior Art

Transcription of genes is performed by RNA polymerase. RNA polymerasesynthesizes ribonucleoside phosphates in the 5′ to 3′ direction usingdouble-stranded DNA as a template in a primer independent manner. In thecase of Escherichia coli, for example, its RNA polymerase takes the formof a holoenzyme in which ρ factor having promoter recognition ability isbound to the core enzyme β′βα₂. This RNA polymerase initiatestranscription and elongates RNA chain; the transcription is terminatedby the binding of ρ factor. On the other hand, in the case ofeucaryotes, RNA polymerase is classified into RNA polymerases I, II andIII, any of which has a complicated structure composed of more than 10subunits. RNA polymerase I selectively transcribes rRNA; RNA polymeraseII selectively transcribes mRNA precursor; and RNA polymerase IIIselectively transcribes tRNA and 5SrRNA. The amount of RNA synthesizedby such RNA polymerase varies widely depending on the growth stage ofthe relevant cells and environmental changes around them. Atranscription factor which positively or negatively regulates thetranscription initiation of RNA polymerase is deeply involved in thevariation in the amount of RNA synthesis.

Generally, living cells are exposed to an external environment composedof a number of factors including temperature, pressure, oxygen, light,radioactive rays, metal ions, organic compounds, etc. When these factorsvary, cells perceive such changes as stress and make characteristicresponses to them. For example, cells exhibit a response called “heatshock response” to high temperatures. From this response, the expressionof a group of heat shock proteins (HSPs) is induced. HSPs prevent theirreversible precipitation of heat-denatured proteins and have thefunction of molecular chaperone that facilitates the refolding of suchproteins, thereby protecting cells from heat stress. It is known that atranscription factor called “heat shock factor (HSF)” plays an importantrole in the manifestation of the above-described heat shock response inhuman, Xenopus, Drosophila, etc. [Kazuhiro Nagata, Cell Technology,10:348-356 (1991)]. When activated by heat shock, HSF binds to heatshock element (HSE) located upstream of a gene coding for HSP (alsoknown as heat shock gene) to thereby promote the transcription of theheat shock gene.

On the other hand, it is also reported that plants induce stressproteins such as LEA proteins, water channel proteins or synthetases forcompatible solutes in their cells when they are exposed to stress suchas dehydration, low temperature, freezing or salt, thereby protectingtheir cells from such stress. However, much more research is required toelucidate transcription factors which regulate the transcription ofgenes encoding those stress proteins.

OBJECTS AND SUMMARY OF THE INVENTION

It is an object of the present invention to provide a protein whichregulates the transcription of genes located downstream of a stressresponsive element which is essential for controlling stress responsivegene expression; a gene encoding the protein; a recombinant vectorcomprising the gene; a transformant comprising the recombinant vector; atransgenic plant comprising the gene; a method for producing the aboveprotein using the transformant; and a method for determining a stresslevel in a plant.

As a result of extensive and intensive researches toward the solution ofthe above-described problem, the present inventors have succeeded inisolating from a low temperature resistant plant Arabidopsis thaliana agene coding for a transcription factor which binds to a stressresponsive element and activates the transcription of genes locateddownstream of the element. Thus, the present invention has beenachieved.

The present invention relates to the following recombinant protein (a)or (b):

(a) a protein consisting of the amino acid sequence as shown in SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10;

(b) a protein which consists of the amino acid sequence having deletion,substitution or addition of at least one amino acid in the amino acidsequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ IDNO: 10 and which regulates the transcription of genes located downstreamof a stress responsive element.

Further, the present invention relates to a transcription factor genecoding for the following protein (a) or (b):

(a) a protein consisting of the amino acid sequence as shown in SEQ IDNO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ ID NO: 10;

(b) a protein which consists of the amino acid sequence having deletion,substitution or addition of at least one amino acid in the amino acidsequence as shown in SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 8 or SEQ IDNO: 10 and which regulates the transcription of genes located downstreamof a stress responsive element.

Further, the present invention relates to a gene comprising thefollowing DNA (c) or (d):

(c) a DNA consisting of the nucleotide sequence as shown in SEQ ID NO:1, SEQ ID NO: 3, SEQ ID NO: 7 or SEQ ID NO: 9;

(d) a DNA which hybridizes with the DNA consisting of the nucleotidesequence as shown in SEQ ID NO: 1, SEQ ID NO: 3, SEQ ID NO: 7 or SEQ IDNO: 9 under stringent conditions and which codes for a protein thatregulates the transcription of genes located downstream of a stressresponsive element.

Specific examples of the above-mentioned stress include dehydrationstress, low temperature stress and salt stress.

Further, the present invention relates to a recombinant vectorcomprising the gene of the invention.

Further, the present invention relates to a transformant comprising therecombinant vector.

Further, the present invention relates to a transgenic plant comprisingthe gene of the invention.

Further, the present invention relates to a method for producing aprotein which regulates the transcription of genes located downstream ofa stress responsive element, comprising culturing the above transformantin a medium and recovering the protein from the resultant culture.

Further, the present invention relates to a method for determining astress level in a plant, comprising determining a transcription level ofthe gene of the invention in the plant.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram showing the principle of screening of the gene ofthe invention.

FIG. 2A shows DRE (dehydration responsive element) region.

FIG. 2B presents photographs showing the results of gel shift assay onthe DRE-binding property of DREB1A and DREB2A proteins.

FIG. 3A depicts construction of an Effector Plasmid and a ReportorPlasmid, respectively.

FIG. 3B presents diagrams showing the transcription activating abilityof DREB 1A and DREB2A proteins.

FIG. 4 is a diagram showing the structure of a recombinant plasmid to beintroduced into a plant.

FIG. 5 presents photographs showing transcription levels of individualgenes in DREB1A gene-introduced plants when stress is given.

FIG. 6 presents photographs showing the growth of DREB2A gene-introducedplants when freezing stress or dehydration stress is given.

DETAILED DESCRIPTION OF THE INVENTION

Hereinbelow, the present invention will be described in detail.

The gene of the invention is a gene encoding a protein (transcriptionfactor) which binds to a cis element located upstream of genes encodingstress responsive proteins expressed in response to environmentalstresses such as low temperature, dehydration or salt, to therebyactivate the transcription of the genes of those stress responsiveproteins. Specific examples of the above cis element include dehydrationresponsive element (DRE), abscisic acid responsive element (ABRE) andlow temperature responsive element. The protein encoded by the gene ofthe invention has a function to activate the transcription of geneslocated downstream of the above-mentioned stress responsive element.

In the present invention, genes encoding DRE-binding proteins will beexplained by way of example. Hereinafter, the genes of the invention arereferred to as “DRE-binding protein 1A gene” (also called “DREB1Agene”), “DRE-binding protein 1C gene” (also called “DREB1C gene”),“DRE-binding protein 2A gene” (also called “DREB2A gene”) and“DRE-binding protein 2B gene” (also called “DREB2B gene”).

1. Cloning of the Gene of the Invention

1-1. Preparation of mRNA and a cDNA Library from Arabidopsis thaliana.

As a source of mRNA, a part of the plant body of Arabidopsis thalianasuch as leaves, stems, roots or flowers, or the plant body as a wholemay be used. Alternatively, plant bodies obtained by sowing seeds ofArabidopsis thaliana on a solid medium such as GM medium, MS medium or#3 medium and growing them aseptically may be used. The mRNA level ofDREB1A gene of the invention in Arabidopsis thaliana plants increaseswhen they are exposed to low temperature stress (e.g. 10 to −4° C.). Onthe other hand, the mRNA level of DREB2A gene of the invention increaseswhen the plants are exposed to salt stress (e.g. 150-250 mM NaCl) ordehydration stress (e.g. dehydrated state). Therefore, Arabidopsisthaliana plants which have been exposed to such stress may also be used.

mRNA is prepared, for example, by exposing Arabidopsis thaliana plantbodies grown on GM medium to low temperature stress, dehydration stressor salt stress and then freeze them with liquid nitrogen. Subsequently,conventional techniques for mRNA preparation may be used. For example,the frozen plant bodies are ground in a mortar. From the resultantground material, crude RNA fraction is extracted by the glyoxal method,the guanidine thiocyanate-cesium chloride method, the lithiumchloride-urea method, the proteinase K-deoxyribonuclease method or thelike. From this crude RNA fraction, poly(A)⁺ RNA (mRNA) can be obtainedby the affinity column method using oligo dT-cellulose or polyU-Sepharose carried on Sepharose 2B or by the batch method. Theresultant mRNA may further be fractionated by sucrose gradientcentrifugation or the like.

Single-stranded cDNA is synthesized using the thus obtained mRNA as atemplate; this synthesis is performed using a commercial kit (e.g.ZAP-cDNA Synthesis Kit: Stratagene), oligo(dT)₂₀ and a reversetranscriptase. Then, double-stranded cDNA is synthesized from theresultant single-stranded cDNA. An appropriate adaptor such asEcoRI-NotI-BamHI adaptor is added to the resultant double-stranded cDNA,which is then ligated downstream of a transcriptional activation domain(such as GAL4 activation domain) in a plasmid (such as pAD-GAL4 plasmid:Stratagene) containing such a domain to thereby prepare a cDNA library.

1-2. A Host to be Used in the Cloning of the Gene of the Invention

The gene of the invention can be cloned, for example, by one hybridscreening using yeast. The screening by this method may be performedusing a commercial kit (e.g. Matchmaker One Hybrid System: Clontech).

In the cloning of the gene of the invention using the above-mentionedkit, first, a DNA comprising DRE sequences to which the transcriptionfactor of the invention binds is ligated to both plasmids pHISi-1 andpLacZi contained in the kit. The thus constructed plasmids aretransformed into the yeast contained in the kit (Saccharomaycescerevisiae YM4271) to thereby prepare a host yeast for cloning.

The host yeast for cloning can biosynthesize histidine by the action ofHIS3 protein which is expressed leakily by HIS3 minimum promoter. Thus,this yeast can survive in the absence of histidine. However, since thepromoter used for the expression of the gene encoding HIS3 protein is aminimum promoter which can only maintain the minimum transcriptionlevel, HIS3 protein produced in cells is extremely small in quantity.Therefore, when the host yeast is cultured in the presence of 3-AT(3-aminotriazole) that is a competitive inhibitor against HIS protein,the function of HIS3 protein in cells is inhibited by 3-AT in aconcentration dependent manner. When the concentration of 3-AT exceeds aspecific level, HIS3 protein in cells becomes unable to function and, asa result, the host yeast becomes unable to grow in the absence ofhistidine.

Since lacZ gene is also located downstream of CYC1 minimum promoter,β-galactosidase is produced only in extremely small quantity in theyeast cells. Thus, when the host yeast is plated on an Xgal containingplate, colonies appearing thereon do not have such Xgal degradingability that turns the colonies into blue as a whole. However, when atranscription factor that binds to DRE located upstream of HIS3 and lacZgenes to activate the transcription thereof is expressed in the hostyeast, the yeast becomes viable in the presence of 3-AT and, at the sametime, Xgal is degraded to turn the colonies into blue.

As used herein, the term “dehydration responsive element (DRE)” refersto a cis-acting DNA domain consisting of a 9 bp conserved sequence5′-TACCGACAT-3′ located upstream of those genes which are expressed uponexposure to dehydration stress, low temperature stress, etc.

A DNA region comprising DRE can be obtained by amplifying the promoterregion (from −215 to −145 based on the translation initiation site) ofrd29A gene [Kazuko Yamaguchi-Shinozaki and Kazuo Shinozaki, The PlantCell 6:251-264 (1994)], one of dehydration tolerance genes, bypolymerase chain reaction (PCR). As a template DNA which can be used inthis PCR, genomic DNA from Arabidopsis thaliana is given. As a senseprimer, 5′-aagcttaagcttacatcagt ttgaaagaaa-3′ (SEQ ID NO: 11) may beused. As an antisense primer, 5′-aagcttaagcttgctttttggaactcatgtc- 3′(SEQ ID NO: 12) may be used. Other primers may also be used in thepresent invention.

1-3. Cloning of DREB2A Gene and DREB2A Gene

DREB2A gene and DREB2A gene of the invention can be obtained bytransforming the cDNA library obtained in subsection 1-1 above into thehost obtained in subsection 1-2 above by the lithium acetate method orthe like, plating the resultant transformant on LB medium plate or thelike containing Xgal (5-bromo-4-chloro-3-indolyl- β-D-galactoside) and3-AT (3-aminotriazole), culturing the transformant, selecting bluecolonies appearing on the plate and isolating the plasmids therefrom.

Briefly, a positive clone containing DREB1A gene or DREB2A gene of theinvention contains a fusion gene composed of a DNA region coding forGAL4 activation domain (GAL4 AD) and a DNA region coding for DRE-bindingprotein, and expresses a fusion protein (hybrid protein) composed ofDRE-binding protein and GAL4 activation domain. Subsequently, theexpressed fusion protein binds, through DRE-binding protein, to DRElocated upstream of a reporter gene. Then, GAL4 activation domainactivates the transcription of lacZ gene and HIS3 gene. As a result, thepositive clone produces remarkable amounts of HIS3 protein andβ-galactosidase. Thus, because of the action of the HIS3 proteinabundantly produced, the positive clone can biosynthesize histidine evenin the presence of 3-AT. Therefore, the clone becomes viable in thepresence of 3-AT and, at the same time, the Xgal in the medium isdegraded by the β-galactosidase produced to turn the colonies into blue.

Subsequently, these colonies are subjected to single cell isolation. Theisolated cells are cultured. Then, plasmid DNA is purified from thecultured cells to thereby obtain DREB1A gene or DREB2A gene of theinvention.

1-4. Homologues to DREB1A Protein or DREB2A Protein

Organisms may have a plurality of genes with similar nucleotidesequences which are considered to have evolved from a single gene.Proteins encoded by such genes are mutually called homologues. They canbe cloned from the relevant gene library using as a probe a part of thegene of which the nucleotide sequence has already been known. In thepresent invention, genes encoding homologues to DREB1A or DREB2A proteincan be cloned from the Arabidopsis thaliana cDNA library using DREB1AcDNA or DREB2A cDNA obtained in subsection 1-3 above as a probe.

1-5. Determination of Nucleotide Sequences

The cDNA portion is cut out from the plasmid obtained in subsection 1-3or 1-4 above using a restriction enzyme and ligated to an appropriateplasmid such as pSK (Stratagene) for sub-cloning. Then, the entirenucleotide sequence is determined. Sequencing can be performed byconventional methods such as the chemical modification method byMaxam-Gilbert or the dideoxynucleotide chain termination method usingM13 phage. Usually, sequencing is carried out with an automated DNAsequencer (e.g. Perkin-Elmer Model 373A DNA Sequencer).

SEQ ID NOS: 1, 3, 7 and 9 show nucleotide sequences for the genes of theinvention, and SEQ ID NOS: 2, 4, 8 and 10 show amino acid sequences forthe proteins of the invention. As long as a protein consisting of one ofthese amino acid sequences has a function to bind to DRE to therebyactivate the transcription of genes located downstream of DRE, the aminoacid sequence may have mutation (such as deletion, substitution oraddition) in at least one amino acid.

For example, at least 1 amino acid, preferably 1 to about 20 aminoacids, more preferably 1 to 5 amino acids may be deleted in the aminoacid sequence shown in SEQ ID NO: 2, 4, 8 or 10; at least 1 amino acid,preferably 1 to about 20 amino acids, more preferably 1 to 5 amino acidsmay be added to the amino acid sequence shown in SEQ ID NO: 2, 4, 8 or10; or at least 1 amino acid, preferably 1 to about 160 amino acids,more preferably 1 to 40 amino acids may be substituted with other aminoacid(s) in the amino acid sequence shown in SEQ ID NO: 2, 4, 8 or 10.

Also, a DNA which can hybridize with the above-mentioned gene understringent conditions is included in the gene of the invention. The“stringent conditions” means, for example, those conditions in whichformamide concentration is 30-50%, preferably 50%, and temperature is37-50° C., preferably 42° C.

The introduction of mutation into the gene of the invention may beperformed by known techniques such as the method of Kunkel, the gappedduplex method or variations thereof using a mutation introducing kit[e.g. Mutant-K (Takara) or Mutant-G (Takara)] utilizing site-directedmutagenesis or using a LA PCR in vitro Mutagenesis Series Kit (Takara).

Once the nucleotide sequence for the gene of the invention has beendetermined definitely, the gene of the invention can be obtained bychemical synthesis, by PCR using the cDNA or genomic DNA of the gene ofthe invention as a template, or by hybridization of a DNA fragmenthaving the above nucleotide sequence as a probe.

The recombinant vectors of the invention were introduced into E coliK-12 strain and deposited at the National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology (1-3,Higashi 1-Chome, Tsukuba City, Ibaraki Pref., Japan) as FERM BP-6654(DREB1A gene-introduced strain) and FERM BP-6655 (DREB2A gene-introducedstrain) on Aug. 11, 1998.

2. Determination of the DRE Binding Ability and Transcription ActivatingAbility of the Proteins of the Invention

2-1. Analysis of the DRE Binding Ability of DREB1A and DREB2A Proteins

The ability of DREB1A or DREB2A protein to bind to DRE can be confirmedby performing a gel shift assay [Urao, T. et al., Plant Cell 5:1529-1539(1993)] using a fusion protein composed of DREB1A or DREB2A protein andGST. The fusion protein composed of DREB1A or DREB2A protein and GST canbe prepared by ligating DREB1A or DREB2A gene downstream of the GST(glutathione-S-transferase) coding region of a plasmid containing GSTgene (e.g. pGEX-4T-1 vector: Pharmacia) so that the reading frames ofthe two genes coincide with each other, transforming the resultantplasmid into E. coli, culturing the E. coli under conditions whichinduce synthesis of the fusion protein and purifying the fusion proteinfrom the resultant culture.

Gel shift assay is a method for examining the interaction between a DNAand a protein. Briefly, DRE-containing DNA fragment labelled with ³²P orthe like is mixed with the fusion protein described above and incubated.The resultant mixture is electrophoresed. After drying, the gel isautoradiographed to detect those bands which have migrated behind as aresult of the binding of the DNA fragment and the protein. In thepresent invention, the specific binding of DREB1A or DREB2A protein tothe DRE sequence can be confirmed by making it clear that theabove-mentioned behind band is not detected when a DNA fragmentcontaining a varied DRE sequence is used.

2-2. Analysis of the Transcription Activating Ability of the Proteins ofthe Invention

The transcription activating ability of the proteins of the inventioncan be analyzed by a trans activation experiment using a protoplastsystem from Arabidopsis thaliana. For example, DREB1A cDNA is ligated topBI221 plasmid (Clontech) containing CaMV35S promoter to construct aneffector plasmid. On the other hand, 3 cassettes of the DRE-containing71 base DNA region obtained in subsection 1-2 above are connectedtandemly to prepare a DNA fragment, which is ligated upstream of TATApromoter located upstream of β-glucuronidase (GUS) gene in pBI221plasmid to construct a reporter plasmid. Subsequently, these twoplasmids are introduced into protoplasts of Arabidopsis thaliana andthen GUS activity is determined. If GUS activity is increased by thesimultaneous expression of DREB1A protein, it is understood that DREB1Aprotein expressed in the protoplasts is activating the transcription ofGUS gene through the DRE sequence.

In the present invention, preparation of protoplasts and introduction ofplasmid DNA into the protoplasts may be performed by the method of Abelet al. [Abel, S. et al., Plant J. 5:421-427 (1994)]. In order tominimize experimental errors resulted from the difference in plasmid DNAintroduction efficiency by experiment, a plasmid in which luciferasegene is ligated downstream of CaMV35S promoter may be introduced toprotoplasts together with the two plasmids described above, andβ-glucuronidase activity against luciferase activity may be determined.Then, the determined value may be taken as a value indicating thetranscription activating ability. β-glucuronidase activity can bedetermined by the method of Jefferson [Jefferson, R. A., EMBO J.83:8447-8451 (1986)]; and luciferase activity can be determined usingPicaGene Luciferase Assay Kit (Toyo Ink).

3. Preparation of Recombinant Vectors and Transformants

3-1. Preparation of Recombinant Vectors

The recombinant vector of the invention can be obtained by ligating(inserting) the gene of the invention to (into) an appropriate vector.The vector into which the gene of the invention is to be inserted is notparticularly limited as long as it is replicable in a host. For example,plasmid DNA, phage DNA or the like may be used.

Specific examples of plasmid DNA include plasmids for E coli hosts suchas pBR322, pBR325, pUC118, pUC119; plasmids for Bacillus subtilis hostssuch as pUB110, pTP5; plasmids for host yeasts such as YEp13, YEp24,YCp50; and plasmids for host plant cells such as pBI221, pBI121.Specific examples of phage DNA include λ phage and the like. Further, ananimal virus vector such as retrovirus or vaccinia virus; or an insectvirus vector such as baculovirus may also be used.

For insertion of the gene of the invention into a vector, a method maybe employed in which the purified DNA is digested with an appropriaterestriction enzyme and then inserted into the restriction site or themulti-cloning site of an appropriate vector DNA for ligation to thevector.

The gene of the invention should be operably incorporated into thevector. For this purpose, the vector of the invention may contain, ifdesired, cis elements (such as enhancer), a splicing signal, poly(A)addition signal, selection marker, ribosome binding sequence (SDsequence) or the like in addition to a promoter and the gene of theinvention. As the selection marker, dihydrofolate reductase gene,ampicillin resistance gene, neomycin resistance gene, or the like may beenumerated.

3-2. Preparation of Transformants

The transformant of the invention can be obtained by introducing therecombinant vector of the invention into a host so that the gene ofinterest can be expressed. The host is not particularly limited as longas it can express the gene of the invention. Specific examples of thehost include Escherichia bacteria such as E coli; Bacillus bacteria suchas Bacillus subtilis; Pseudomonas bacteria such as Pseudomonas putida;Rhizobium bacteria such as Rhizobium meliloti; yeasts such asSaccharomyces cerevisiae, Schizosaccharomyces pombe; plant cell strainsestablished from Arabidopsis thaliana, tobacco, maize, rice, carrot,etc. or protoplasts prepared from such plants; animal cells such as COScells, CHO cells; or insect cells such as Sf9 cells, Sf21 cells.

When a bacterium such as E coli is used as the host, the recombinantvector of the invention is capable of autonomous replication in the hostand, at the same time, it is preferably composed of a promoter, aribosome binding sequence, the gene of the invention and a transcriptiontermination sequence. The vector may also contain a gene to control thepromoter.

As E coli, HMS174 (DE3), K12 or DH1 strain may be used, for example. AsBacillus subtilis, MI 114 or 207-21 strain may be used, for example.

As the promoter, any promoter may be used as long as it can direct theexpression of the gene of interest in a host such as E. coli. Forexample, an E coli- or phage-derived promoter such as trp promoter, lacpromoter, P_(L) promoter or P_(R) promoter may be used. An artificiallydesigned and altered promoter such as tac promoter may also be used.

As a method for introducing the recombinant vector into a bacterium, anymethod of DNA introduction into bacteria may be used. For example, amethod using calcium ions [Cohen, S. N. et al., Proc. Natl. Acad. Sci.,USA, 69:2110-2114 (1972)], electroporation, or the like may be used.

When a yeast is used as the host, Saccharomyces cerevisiae,Schizosaccharomyces pombe, Pichia pastoris or the like is used. In thiscase, the promoter to be used is not particularly limited. Any promotermay be used as long as it can direct the expression of the gene ofinterest in yeast. For example, gal1 promoter, gal10 promoter, heatshock protein promoter, MFα1 promoter, PH05 promoter, PGK promoter, GAPpromoter, ADH promoter, AOX1 promoter or the like may be enumerated.

As a method for introducing the recombinant vector into yeast, anymethod of DNA introduction into yeast may be used. For example,electroporation [Becker, D. M. et al., Methods Enzymol., 194:182-187(1990)], the spheroplast method [Hinnen, A. et al., Proc. Natl. Acad.Sci., USA, 75:1929-1933 (1978)], the lithium acetate method [Itoh, H.,J. Bacteriol., 153:163-168 (1983)] or the like may be enumerated.

When a plant cell is used as the host, a cell strain established fromArabidopsis thaliana, tobacco, maize, rice, carrot, etc. or a protoplastprepared from such plants may be used. In this case, the promoter to beused is not particularly limited as long as it can direct the expressionof the gene of interest in plants. For example, 35S RNA promoter ofcauliflower mosaic virus, rd29A gene promoter, rbcS promoter or the likemay be enumerated.

As a method for introducing the recombinant vector into a plant, themethod of Abel et al. using polyethylene glycol [Abel, H. et al., PlantJ. 5:421-427 (1994)], electroporation or the like may be used.

When an animal cell is used as the host, simian COS-7 or Vero cells,Chinese hamster ovary cells (CHO cells), mouse L cells, rat GH3 cells,human FL cells or the like may be used. As a promoter, SRα promoter,SV40 promoter, LTR promoter, CMV promoter or the like may be used. Theearly gene promoter of human cytomegalovirus may also be used.

As a method for introducing the recombinant vector into an animal cell,electroporation, the calcium phosphate method, lipofection or the likemay be enumerated.

When an insect cell is used as the host, Sf9 cells, Sf21 cells or thelike may be used.

As a method for introducing the recombinant vector into an insect cell,the calcium phosphate method, lipofection, electroporation or the likemay be used.

4. Production of the Proteins of the Invention

The protein of the invention is a protein having the amino acid sequenceencoded by the gene of the invention; or a protein which has the aboveamino acid sequence having the mutation described above at least at oneamino acid and yet which has a function to regulate the transcription ofgenes located downstream of a stress responsive element. In thisspecification, the protein encoded by DREB1A gene is called “DREB1Aprotein”; the protein encoded by DREB1B gene is called “DREB1B protein”;the protein encoded by DREB1C gene is called “DREB1C protein”; theprotein encoded by DREB2A gene is called “DREB2A protein”; and theprotein encoded by DREB2B gene is called “DREB2B protein”.

The protein of the invention can be obtained by culturing thetransformant described above in a medium and recovering the protein fromthe resultant culture. The term “culture” means any of the followingmaterials: culture supernatant, cultured cells, cultured microorganisms,or disrupted cells or microorganisms.

The cultivation of the transformant of the invention in a medium iscarried out by conventional methods used for culturing a host.

As a medium for culturing the transformant obtained from a microorganismhost such as E. coli or yeast, either a natural or synthetic medium maybe used as long as it contains carbon sources, nitrogen sources andinorganic salts assimilable by the microorganism and is capable ofefficient cultivation of the transformant. When a plant cell is used asthe host, vitamins such as thiamine and pyridoxine are added to themedium if necessary. When an animal cell is used as the host, a serumsuch as RPMI1640 is added to the medium if necessary.

As carbon sources, carbohydrates such as glucose, fructose, sucrose,starch; organic acids such as acetic acid, propionic acid; and alcoholssuch as ethanol and propanol may be used.

As nitrogen sources, ammonia; ammonium salts of inorganic or organicacids such as ammonium chloride, ammonium sulfate, ammonium acetate,ammonium phosphate; other nitrogen-containing compounds; Peptone; meatextract; corn steep liquor and the like may be used.

As inorganic substances, potassium dihydrogen phosphate, dipotassiumhydrogen phosphate, magnesium phosphate, magnesium sulfate, sodiumchloride, iron(II) sulfate, manganese sulfate, copper sulfate, calciumcarbonate and the like may be used.

Usually, the cultivation is carried out under aerobic conditions (suchas shaking culture or aeration agitation culture) at 37° C. for 6 to 24hrs. During the cultivation, the pH is maintained at 7.0 to 7.5. The pHadjustment is carried out with an inorganic or organic acid, an alkalisolution or the like.

During the cultivation, an antibiotic such as ampicillin or tetracyclinemay be added to the medium if necessary.

When a microorganism transformed with an expression vector containing aninducible promoter is cultured, an inducer may be added to the medium ifnecessary. For example, when a microorganism transformed with anexpression vector containing Lac promoter is cultured,isopropyl-β-D-thiogalactopyranoside (IPTG) or the like may be added.When a microorganism transformed with an expression vector containingtrp promoter is cultured, indoleacrylic acid (IAA) or the like may beadded.

Usually, the cultivation of such a microorganism is carried out in thepresence 5% CO₂ at 37° C. for 1 to 30 days. During the cultivation, anantibiotic such as kanamycin or penicillin may be added to the medium ifnecessary.

After the cultivation, the protein of the invention is extracted bydisrupting the cultured microorganisms or cells if the protein isproduced in the microorganisms or cells. If the protein of the inventionis produced outside of the microorganisms or cells, the culture fluid isused as it is or subjected to centrifugation to remove themicroorganisms or cells. Thereafter, the resultant supernatant issubjected to conventional biochemical techniques used forisolating/purifying a protein. These techniques include ammonium sulfateprecipitation, gel chromatography, ion exchange chromatography andaffinity chromatography; these techniques may be used independently orin an appropriate combination to thereby isolate and purify the proteinof the invention from the above culture.

5. Creation of Transgenic Plants into which the Gene of the Invention isIntroduced

A transgenic plant resistant to environmental stresses, in particular,low temperature stress, freezing stress and dehydration stress, can becreated by introducing a DNA encoding the protein of the invention intoa host plant using recombinant techniques. As a method for introducingthe gene of the invention into a host plant, indirect introduction suchas the Agrobacterium infection method, or direct introduction such asthe particle gun method, polyethylene glycol method, liposome method,microinjection method or the like may be used. When the Agrobacteriuminfection method is used, the transgenic plant of the invention can becreated by the following procedures.

5-1. Preparation of a Recombinant Vector to be Introduced into a Plantand Transformation of Agrobacterium

A recombinant vector to be introduced into a plant can be prepared bydigesting with an appropriate restriction enzyme a DNA comprisingDREB1A, DREB1C, DREB2A or DREB2B gene obtained in section 1 above,ligating an appropriate linker to the resultant DNA if necessary, andinserting the DNA into a cloning vector for plant cells. As the cloningvector, a binary vector type plasmid such as pBI2113Not, pBI2113,pBI101, pBI121, pGA482, pGAH, pBIG; or an intermediate vector typeplasmid such as pLGV23Neo, pNCAT, pMON200 may be used.

When a binary vector type plasmid is used, the gene of interest isinserted between the border sequences (LB, RB) of the binary vector. Theresultant recombinant vector is amplified in E. coli. The amplifiedrecombinant vector is introduced into Agrobacterium tumefaciens C58,LBA4404, EHA101, C58C1Rif^(R), EHA105, etc. by freeze-thawing,electroporation or the like. The resultant Agrobacterium tumefaciens isused for the transformation of a plant of interest.

In addition to the method described above, the three-member conjugationmethod [Nucleic Acids Research, 12:8711 (1984)] may also be used toprepare an Agrobacterium containing the gene of the invention for use inplant infection. Briefly, an E. coli containing a plasmid comprising thegene of interest, an E. coli containing a helper plasmid (e.g. pRK2013)and an Agrobacterium are mixed and cultured on a medium containingrifampicin and kanamycin. Thus, a zygote Agrobacterium for infectingplants can be obtained.

For the expression of a foreign gene in a plant body, a promoter and aterminator for plants should be located before and after the structuralgene of the foreign gene, respectively. Specific examples of promoterswhich may be utilized in the present invention include cauliflowermosaic virus (CaMV)-derived 35S transcript [Jefferson, R. A. et al., TheEMBO J. 6:3901-3907 (1987)]; the promoter for maize ubiquitin gene[Christensen, A. H. et al., Plant Mol. Biol. 18:675-689 (1992)]; thepromoter for nopaline synthase (NOS) gene and the promoter for octopin(OCT) synthase gene. Specific examples of useful terminator sequencesinclude CaMV-derived terminator and NOS-derived terminator. Otherpromoters and terminators which are known to function in plant bodiesmay also be used in the present invention.

If the promoter used in a transgenic plant is a promoter responsible forthe constitutive expression of the gene of interest (e.g. CaMV 35Spromoter) and the use thereof has brought about delay in the growth ofthe transgenic plant or dwarfing of the plant, a promoter which directstransient expression of the gene of interest (e.g. rd29A gene promoter)may be used.

If necessary, an intron sequence which enhances the expression of thegene of the invention may be located between the promoter sequence andthe gene. For example, the intron from maize alcohol dehydrogenase(Adh1) [Genes & Development 1:1183-1200 (1987)] may be introduced.

In order to select transformed cells of interest efficiently, it ispreferable to use an effective selection marker gene in combination withthe gene of interest. As the selection marker, one or more genesselected from kanamycin resistance gene (NPTII), hygromycinphosphotransferase gene (htp) which confers resistance to the antibiotichygromycin on plants, phosphinothricin acetyl transferase gene (bar)which confers resistance to bialaphos and the like.

The gene of the invention and the selection marker gene may beincorporated together into a single vector. Alternatively, the two genesmay be incorporated into separate vectors to prepare two recombinantDNAs.

5-2. Introduction of the Gene of the Invention into a Host Plant

In the present invention, the term “host plant” means any of thefollowing: cultured plant cells, the entire plant body of a culturedplant, plant organs (such as leaves, petals, stems, roots, rhizomes,seeds), or plant tissues (such as epidermis, phloem, parenchyma, xylem,vascular bundle). Specific examples of plants which may be used as ahost include Arabidopsis thaliana, tobacco, rice and maize.

When a cultured plant cell, plant body, plant organ or plant tissue isused as a host plant, a DNA encoding the protein of the invention isincorporated into a vector, which is then introduced into plant sectionsby the Agrobacterium infection method, particle gun method orpolyethylene glycol method to thereby transform the host plant.Alternatively, the DNA may be directly introduced to protoplasts byelectroporation to thereby create a transformed plant.

If the gene of interest is introduced by the Agrobacterium infectionmethod, a step of infecting the plant with an Agrobacterium containing aplasmid comprising the gene of interest is essential. This step can beperformed by the vacuum infiltration method [CR Acad. Sci. Paris, LifeScience, 316:1194 (1993)]. Briefly, Arabidopsis thaliana is grown in asoil composed of vermiculite and perlite (50:50). The resultant plant isdipped directly in a culture fluid of an Agrobacterium containing aplasmid comprising the gene of the invention, placed in a desiccator andthen sucked with a vacuum pump to 65-70 mmHg. Then, the plant wasallowed to stand at room temperature for 5-10 min. The plant pot istransferred to a tray, which is covered with a wrap to maintain thehumidity. The next day, the wrap is removed. The plant is grown in thatstate to harvest seeds.

Subsequently, the seeds are sown on MS agar medium supplemented withappropriate antibiotics to select those individuals which have the geneof interest. Arabidopsis thaliana grown on this medium are transferredto pots and grown there. As a result, seeds of a transgenic plant intowhich the gene of the invention is introduced can be obtained.

Generally, introduced genes are located on the genome of the host plantin a similar manner. However, due to the difference in the locations onthe genome, the expression of the introduced genes varies. This is aphenomenon called position effect. By assaying transformants by Northernblotting with a DNA fragment from the introduced gene as a probe, it ispossible to select those transformants in which the introduced gene isexpressed more highly.

The confirmation that the gene of interest is integrated in thetransgenic plant of the invention and in the subsequent generationthereof can be made by extracting DNA from cells and tissues of thoseplants by conventional methods and detecting the introduced gene by PCRor Southern analysis known in the art.

5-3. Analysis of the Expression Level and Expression Site of the Gene ofthe Invention in Plant Tissues

The expression level and expression site of the gene of the invention ina transgenic plant into which the gene is introduced can be analysed byextracting RNA from cells and tissues of the plant by conventionalmethods and detecting the mRNA of the introduced gene by RT-PCR orNorthern analysis known in the art. Alternatively, the expression leveland expression site can be analysed directly by Western blotting or thelike of the product of the gene of the invention using an antibodyraised against the above product.

5-4. Changes in the mRNA Levels of Various Genes in a Transgenic Plantinto which the Gene of the Invention is Introduced

It is possible to identify by Northern blot analysis those genes whoseexpression levels are believed to have been changed as a result of theaction of the transcription factor of the invention in a transgenicplant into which the gene of the invention is introduced. Northernblotting can assay those genes by comparing their expression in thetransgenic plant into which the gene of the invention is introduced andin plants into which the gene is not introduced.

For example, plants grown on GM agar medium or the like are givendehydration and/or low temperature stress for a specific period of time(e.g. 1 to 2 weeks). Dehydration stress may be given by pulling out theplant from the agar medium and drying it on a filter paper for 10 min to24 hr. Low temperature stress may be given by retaining the plant at 15to −4 ° C. for 10 min to 24 hr. Total RNA is prepared from controlplants which did not receive any stress and plants which receiveddehydration and low temperature stresses. The resultant total RNA issubjected to electrophoresis. Then, genes expressing are assayed byNorthern blot analysis or RT-PCR.

5-5. Evaluation of the Tolerance to Environmental Stresses of theTransgenic Plant

The tolerance to environmental stresses of the transgenic plant of theinvention can be evaluated by setting the plant in a pot containing asoil comprising vermiculite, perlite and the like, exposing the plant tovarious stresses such as dehydration, low temperature and freezing, andexamining the survival of the plant. For example, tolerance todehydration stress can be evaluated by leaving the plant without givingwater for 2 to 4 weeks and then examining the survival. Tolerance tofreezing stress can be evaluated by leaving the plant at −6 to −10° C.for 5 to 10 days, growing it at 20 to 25° C. for 5 to 10 days and thenexamining its survival ratio.

6. Antibodies against the Proteins of the Invention

In the present invention, antibodies against the proteins of theinvention can also be prepared. The term “antibody” means an antibodymolecule as a whole or a fragment thereof (e.g. Fab or F(ab′)₂ fragment)which can bind to the protein of the invention that is an antigen. Theantibody may be either polyclonal or monoclonal.

The antibody against the protein of the invention may be prepared byvarious methods. Such methods of antibody preparation are well known inthe art [see, for example, Sambrook, J. et al., Molecular Cloning, ColdSpring Harbor Laboratory Press (1989)].

6-1. Preparation of Polyclonal Antibodies against the Proteins of theInvention

One of the proteins of the invention genetically engineered as describedabove or a fragment thereof is administered as an antigen to a mammalsuch as rat, mouse or rabbit. The dosage of the antigen per animal is100 to 200 μg when an adjuvant is used. As the adjuvant, Freund'scomplete adjuvant (FCA), Freund's incomplete adjuvant (FIA), aluminumhydroxide adjuvant or the like may be used. Immunization is performedmainly by intravenous, subcutaneous or intraperitoneal injection. Theinterval of immunization is not particularly limited; immunization iscarried out 1 to 5 times, preferably 5 times, at intervals of severaldays to several weeks, preferably at intervals of one week.Subsequently, 7 to 10 days after the final immunization, antibody titeris determined by enzyme immunoassay (EIA), radioimmunoassay (RIA) or thelike. Blood is collected from the animal on the day when the maximumantibody titer is shown, to thereby obtain antiserum. When purificationof antibody from the antiserum is necessary, the antibody can bepurified by appropriately selecting or combining conventional methodssuch as ammonium sulfate salting out, ion exchange chromatography, gelfiltration and affinity chromatography.

6-2. Preparation of Monoclonal Antibodies against the Proteins of theInvention

(i) Recovery of Antibody-Producing Cells

One of the proteins of the invention genetically engineered or afragment thereof is administered as an antigen to a mammal such as rat,mouse or rabbit, as described above. The dosage of the antigen peranimal is 100 to 200 μg when an adjuvant is used. As the adjuvant,Freund's complete adjuvant (FCA), Freund's incomplete adjuvant (FIA),aluminum hydroxide adjuvant or the like may be used. Immunization isperformed mainly by intravenous, subcutaneous or intraperitonealinjection. The interval of immunization is not particularly limited;immunization is carried out 1 to 5 times, preferably 5 times, atintervals of several days to several weeks, preferably at intervals of 1to 2 weeks. Subsequently, 7 to 10 days after the final immunization,preferably 7 days after the final immunization, antibody producing cellsare collected. As antibody-producing cells, spleen cells, lymph nodecells, peripheral blood cells, etc. may be enumerated. Among them,spleen cells and local lymph node cells are preferable.

(ii) Cell Fusion

In order to obtain hybridomas, cell fusion between antibody-producingcells and myeloma cells is performed. As the myeloma cells to be fusedto the antibody-producing cells, a commonly available cell strain of ananimal such as mouse may be used. Preferably, a cell strain to be usedfor this purpose is one which has drug selectivity, cannot survive inHAT selective medium (containing hypoxanthine, aminopterin andthymidine) when unfused, and can survive there only when fused toantibody-producing cells. As the myeloma cells, mouse myeloma cellstrains such as P3X63-Ag.8.U1(P3U1), Sp2/0 and NS-1 may be enumerated.

Subsequently, the myeloma cells and the antibody-producing cellsdescribed above are fused. Briefly, the antibody-producing cells (2×10⁷cells/ml) and the myeloma cells (1×10⁷ cells/ml) are mixed in equalvolumes and reacted in the presence of a cell fusion promoter. As thecell fusion promoter, polyethylene glycol with a mean molecular weightof 1,500 Da may be used, for example. Alternatively, theantibody-producing cells and the myeloma cells may be fused in acommercial cell fusion apparatus utilizing electric stimulation (e.g.electroporation).

(iii) Selection and Cloning of a Hybridoma

A hybridoma of interest is selected from the cells after the cellfusion. As a method for this selection, the resultant cell suspension isappropriately diluted with fetal bovine serum-containing RPMI-1640medium or the like and then plated on microtiter plates at a density ofabout 0.8 to 1 cell/well. Then, a selective medium is added to eachwell. Subsequently, the cells are cultured while appropriatelyexchanging the selective medium. As a result, about 10 days after thestart of cultivation in the selective medium, the growing cells can beobtained as hybridomas.

Subsequently, screening is performed as to whether the antibody ofinterest is present in the culture supernatant of the grown hybridomas.The screening of hybridomas may be performed by any of conventionalmethods. For example, a part of the culture supernatant of a well inwhich a hybridoma is grown is collected and subjected to enzymeimmunoassay or radioimmunoassay.

Cloning of the fused cell is performed by the limiting dilution method,for example. Finally, the hybridoma of interest which is a monoclonalantibody-producing cell is established.

(iv) Recovery of the Monoclonal Antibody

As a method for recovering the monoclonal antibody from the thusestablished hybridoma, the conventional cell culture method or abdominaldropsy formation method may be employed.

In the cell culture method, the hybridoma is cultured in an animal cellculture medium such as 10% fetal bovine serum-containing RPMI-1640medium, MEM medium or a serum-free medium under conventional cultureconditions (e.g. at 37° C. under 5% CO₂) for 7 to 14 days. Then, themonoclonal antibody is recovered from the culture supernatant.

In the abdominal dropsy formation method, about 1×10⁷ cells of thehybridoma is administered to the abdominal cavity of an animal syngeneicto the mammal from which the myeloma cells were derived, to therebypropagate the hybridoma greatly. One to two weeks thereafter, theabdominal dropsy or serum is collected.

If purification of the antibody is necessary in the above-mentionedmethod of recovery, the antibody can be purified by appropriatelyselecting or combining conventional methods such as ammonium sulfatesalting out, ion exchange chromatography, gel filtration and affinitychromatography.

Once the polyclonal or monoclonal antibody is thus obtained, theantibody may be bound to a solid carrier as a ligand to thereby preparean affinity chromatography column. With this column, the protein of theinvention can be purified from the above-mentioned source or othersources. Besides, these antibodies can also be used in Western blottingto detect the protein of the invention.

7. Determination of Stress Levels in Plants

The transcription of DREB1A gene of the invention is activated mainly bylow temperature stress, and the transcription of DREB2A gene bydehydration stress and salt stress. Therefore, by determining thetranscription level of the gene of the invention, it is possible to knowthe level of stress such as low temperature, dehydration or salt which aplant is undergoing.

In protected culture of a crop using vinyl houses or the like, theenvironmental arrangement cost for providing light, heat, water, soil,etc. occupies 20-80% of the production cost of the crop. Under suchcircumstances, if it is possible to grasp promptly whether the crop issubjected to low temperature stress, dehydration stress or salt stress,the environmental arrangement cost can be minimized to thereby reducethe production cost greatly.

The transcription level of the gene of the invention can be determinedby RNA gel blot analysis or quantitative PCR, for example. As a probe tobe used in RNA gel blot analysis, DREB1A gene and/or a 100-1000 bp DNAregion comprising a DREB1A gene specific sequence adjacent to DREB1Agene may be used for the detection of DREB1A gene. For the detection ofDREB2A gene, DREB2A gene and/or a 100-1000 bp DNA region comprising aDREB2A gene specific sequence adjacent to DREB2A gene may be used. As aprimer to be used in quantitative PCR, a 17-25 bp oligonucleotide withinthe coding sequence of DREB1A gene or adjacent thereto which is capableof specifically amplifying DREB1A gene may be used for amplifying DREB1Agene. Likewise, a 17-25 bp oligonucleotide within the coding sequence ofDREB2A gene or adjacent thereto which is capable of specificallyamplifying DREB2A gene may be used for amplifying DREB2A gene.

The above-described probe or primer may be used in a kit for determiningthe transcription level of DREB1A or DREB2A gene.

PREFERRED EMBODIMENTS OF THE INVENTION

Hereinbelow, the present invention will be described more specificallywith reference to the following Examples. However, the technical scopeof the present invention is not limited to these Examples.

EXAMPLE 1 Cultivation of Arabidopsis thaliana Plant Bodies

Arabidopsis thaliana seeds obtained from LEHLE were sterilized in asolution containing 1% sodium hypochlorite and 0.02% Triton X-100 for 15min. After rinsing with sterilized water, 40-120 seeds were sown on GMagar medium [4.6 g/L mixed salts for Murashige-Skoog medium (NihonPharmaceutical Co., Ltd.), 0.5 g/L MES, 30 g/L sucrose, 8 g/L agar, pH5.7] and cultured at 22° C. under light conditions of about 1000 lux and16 hr light 8 hr dark, to thereby obtain plant bodies.

EXAMPLE 2 Cloning of DREB1A Gene and DREB2A Gene

(1) Preparation of Poly(A)⁺ RNA

The plant bodies obtained in Example 1 were subjected to low temperaturetreatment at 4° C. for 24 hr, and then total RNA was prepared from themby the glyoxal method. Briefly, 3 g of Arabidopsis thaliana plant bodiesfrozen in liquid nitrogen was suspended in 100 ml of 5.5 M GTC solution(5.5 M guanidine thiocyanate, 25 mM sodium citrate, 0.5% sodiumN-lauroyl sarcosinate) and solubilized quickly with a homogenizer. Thishomogenate was sucked into and extruded from a syringe provided with a18-G needle repeatedly more than 10 times to thereby disrupt the DNA.Then, the homogenate was centrifuged at 4° C. at 12,000×g for 15 min toprecipitate and remove the cell debris.

The resultant supernatant was overlayered on a cushion of 17 ml of CsTFAsolution [a solution obtained by mixing cesium trifluoroacetate(Pharmacia), 0.25 M EDTA and sterilized water to give D=1.51] placed inan autoclaved centrifuge tube, and then ultracentrifuged in BeckmannSW28 Rotor at 15° C. at 25,000×rpm for 24 hr to precipitate total RNA.

The resultant total RNA was dissolved in 600 μl of 4 M GTC solution(obtained by diluting the above-described 5.5 M GTC solution withsterilized water to give a GTC concentration of 4 M) and precipitatedwith ethanol to thereby obtain total RNA.

The resultant total RNA was dissolved in 2 ml of TE/NaCl solution (1:1mixture of TE and 1 M NaCl) and passed through an oligo-dT cellulosecolumn [prepared by packing a Bio-Rad Econocolumn (0.6 cm in diameter)with oligo-dT cellulose (type 3) (Collaborative Research) to a height of1.5 cm] equilibrated with TE/NaCl in advance. The solution passedthrough the column was fed to the column again. Subsequently, the columnwas washed with about 8 ml of TE/NaCl. TE was added thereto to elute andpurify poly(A)⁺ RNA. The amount of the thus obtained RNA was determinedwith a UV spectroscope.

(2) Synthesis of a cDNA Library

Double-stranded cDNA was synthesized with a cDNA synthesis kit(Stratagene) using 5 μg of the poly(A)⁺ RNA obtained in (1) above. Then,the double-stranded cDNA was ligated to pAD-GAL4 plasmid (Stratagene) tothereby synthesize a cDNA library. Briefly, at first, single-strandedcDNA was synthesized in the following reaction solution according to theprotocol attached to the kit.

Poly (A)⁺ RNA  5 μl (5 μg) 10x 1st Strand synthesis buffer  5 μlDEPC-treated water 34 μl 40 U/μl Ribonuclease inhibitor  1 μl Nucleotidemix for 1st strand  3 μl 1.4 μg/μl Linker primer  2 μl Total 50 μl

To the above solution, 1.5 μl (50 U/μl) of reverse transcriptase wasadded and incubated at 37° C. for 1 hr to thereby synthesizesingle-stranded cDNA. To the resultant reaction solution containingsingle-stranded cDNA, the following reagents were added in the indicatedorder.

Reaction solution containing single-stranded cDNA 45 μl 10x 2nd Strandsynthesis buffer 20 μl NTP mix for 2nd strand  6 μl 1.5 U/μl RNase H  2μl 9 U/μl DNA polymerase I 11 μl DEPC-treated water 116 μl  Total 200μl 

The resultant reaction solution was incubated at 16° C. for 2.5 hr tothereby synthesize double-stranded cDNA.

The synthesized double-stranded cDNA was blunt-ended by incubating itwith 5 units of Pfu DNA polymerase at 72° C. for 30 min. Subsequently,the resultant cDNA was subjected to phenol/chloroform extraction andethanol precipitation. To the resultant pellet, 9 μl of EcoRI-NotI-BamHIadaptor (Takara), 1 μl of 10×ligase buffer, 1 μl of ATP and 1 μl of T4DNA ligase (4 U/μl ) were added and incubated at 4° C. for 2 days tothereby add the adaptor to the double-stranded cDNA.

Subsequently, the cDNA having an EcoRI restriction enzyme site at bothends was ligated to the EcoRI site downstream of the GAL4 activationdomain of pAD-GAL4 plasmid (Stratagene) (a cloning vector) with T4 DNAligase to thereby synthesize a cDNA library.

(3) Preparation of Genomic DNA

Genomic DNA was prepared from the plant bodies obtained in Example 1according to the method described by Maniatis, T. et al. [MolecularCloning: A Laboratory Manual, pp. 187-198, Cold Spring Harbor LaboratoryPress, Cold Spring Harbor, N.Y. (1982)]. Briefly, 2,000 ml of disruptionbuffer [0.35 M sucrose, 1 M Tris-HCl (pH 8.0), 5 mM MgCl₂, 50 mM KCl]was added to 50 g of Arabidopsis thaliana plant bodies. The mixture wasdisrupted in a whirling blender for 1 min 3 times to homogenize theplant bodies.

The disrupted solution was filtered to remove the cell residue. Thefiltrate was transferred into centrifuge tubes and centrifuged in aswing rotor at 3,000×g at 4° C. for 10 min at a low speed. The resultantsupernatant was discarded. The precipitate was suspended in 30 ml ofice-cooled disruption buffer and then re-centrifuged at a low speed. Thesame procedures were repeated 3 times until the green precipitate turnedinto white.

The resultant white precipitate was suspended in 10 ml of ice-cooled TE.To this suspension, 10 ml of lysis solution (0.2 M Tris-HCl (pH 8.0), 50mM EDTA, 2% sodium N-lauroyl sarcosinate) was added. Then, 0.1 ml ofproteinase K (10 mg/ml) was added thereto to digest nuclei. Theresultant digest was subjected to phenol treatment and ethanolprecipitation. The DNA fiber obtained by the precipitation was recoveredby centrifugation at 3,000×g for 5 min and dissolved in 1 ml of TE tothereby obtain genomic DNA.

(4) Construction of a Host Yeast for Use in Yeast One Hybrid Screening

For the cloning of a gene encoding the transcription factor (DRE-bindingprotein) of the invention, a host was constructed (FIG. 1). This hostfor cloning comprises two plasmids, one containing 4 cassettes of DREmotif-containing DNA upstream of HIS3 reporter gene and the othercontaining 4 cassettes of DRE motif-containing DNA upstream of lacZreporter gene. Briefly, first, the promoter region of rd29A gene (theregion from −215 to −145 based on the translation initiation site ofrd29A gene) comprising DRE sequence to which the transcription factor ofthe invention binds to was amplified by PCR. As a sense primer,5′-aagcttaagcttacatcagtttgaaagaaa-3′ (SEQ ID NO: 11) was synthesized. Asan antisense primer, 5′-aagcttaagcttgctttttggaactcatgtc-3′ (SEQ ID NO:12) was synthesized. To these primers, a HindIII restriction site wasintroduced to their 5′ end so that PCR fragments can be ligated to avector easily after amplification. These primers were synthesizedchemically with a fully automated DNA synthesizer (Perkin-Elmer). A PCRwas performed using these primers and the genomic DNA from (3) above asa template. The composition of the PCR reaction solution was as follows.

Genomic DNA solution  5 μl (100 ng) Sterilized water 37 μl 10x PCRbuffer [1.2 M Tris-HCl (pH 8.0),  5 μl 100 mM KCl, 60 mM (NH₄)₂SO₄, 1%Triton X-100, 0.1 mg/ml BSA] 50 pmol/μl Sense primer  1 μl (50 pmol) 50pmo1/μl Antisense primer  1 μl (50 pmol) KOD DNA polymerase (KOD-101,TOYOBO)  1 μl (2.5 U) Total 50 μl

After the above reaction solution was mixed thoroughly, 50 μl of mineraloil was overlayered on it. The PCR was performed 25 cycles, one cycleconsisting of thermal denaturation at 98° C. for 15 sec, annealing at65° C. for 2 sec and extension at 74° C. for 30 sec. After completion ofthe reaction, 50 μl of chloroform was added to the reaction solution,and then the resultant mixture was centrifuged at 4° C. at 15,000 rpmfor 15 min. The resultant upper layer was recovered into a freshmicrotube, to which 100 μl of ethanol was added and mixed well. Themixture was centrifuged at 4° C. at 15,000 rpm for 15 min to pellet thePCR product.

The resultant PCR product was digested with HindIII and then ligated tothe HindIII site of vector pSK to yield a recombinant plasmid. Thisplasmid was transformed into E coli. From the transformant, plasmid DNAwas prepared to determine the nucleotide sequence. By these procedures,a transformant comprising pSK with a DNA fragment containing 4 cassettesof DRE connected in the same direction was selected.

The DNA fragment containing 4 cassettes of DRE was cut out from pSKplasmid using EcoRI and HincII, and then ligated to the EcoRI-MluI siteupstream of the HIS3 minimum promoter of a yeast expression vectorpHISi-1 (Clontech). Likewise, the DRE-containing DNA fragment was cutout from pSK plasmid using EcoRI and HincII, and then ligated to theEcoRI-SalI site upstream of the lacZ minimum promoter of a yeastexpression vector pLacZi (Clontech). The resultant two plasmids weretransformed into Saccharomyces cerevisiae YM4271 (MATa, ura3-52,his3-200, ade2-101, lys2-801, leu2-3, 112, trp1-903) (Clontech) tothereby yield a host yeast to be used in yeast one hybrid screening(FIG. 1).

(5) Cloning of DREB1A Gene and DREB2A Gene

The host yeast prepared in (4) above was transformed with the cDNAlibrary prepared in (2) above. The resultant yeast transformants(1.2×10⁶) were cultured and screened as described previously. As aresult, two positive clones were obtained. The cDNAs of these cloneswere cut out from pAD-GAL4 plasmid using EcoRI and then ligated to theEcoRI site of pSK plasmid to thereby obtain pSKDREB1A and pSKDREB2A.

(6) Determination of the Nucleotide Sequences

The entire nucleotide sequences were determined on plasmids pSKDREB1Aand pSKDREB2A. The plasmids used for the sequencing were prepared withan automated plasmid preparation apparatus Model PI-100 (Kurabo). Forthe sequencing reaction, a reaction robot CATALYST 800 (Perkin Elmer)was used. For the DNA sequencing, Perkin Elmer Sequencer Model 373A wasused. As a result, it was found that the cDNA from plasmid pSKDREB1Aconsists of 933 bases. From the analysis of its open reading frame, itwas found that the gene product encoded by DREB1A gene is a proteinconsisting of 216 amino acid residues with a molecular weight of about24.2 kDa. This protein is encoded by the nucleotide sequence fromposition 119 (adenine) to position 766 (thymine) of SEQ ID NO: 1. On theother hand, it was found that the cDNA from plasmid pSKDREB2A consistsof 1437 bases. From the analysis of its open reading frame, it was foundthat the gene product encoded by DREB2A gene is a protein consisting of335 amino acid residues with a molecular weight of about 37.7 kDa.

(7) Isolation of Genes Encoding Homologues to DREB1A or DREB2A Protein

Genes encoding homologues to the protein encoded by DREB1A or DREB2Agene were isolated. Briefly, genes encoding such homologues wereisolated from Arabidopsis thaliana λgt11 cDNA library using as a probethe double-stranded cDNA fragment comprising DREB1A or DREB2A geneobtained in (5) above according to the method described by Sambrook, J.et al., Molecular Cloning: A Laboratory Manual 2nd Ed., Cold SpringHarbor Laboratory Press, N.Y. (1989)]. As genes encoding homologues toDREB1A protein, DREB1B gene and DREB1C gene were obtained; as a geneencoding a homologue to DREB2A protein, DREB2B gene was obtained. As aresult of DNA sequencing, it was found that DREB1B gene (SEQ ID NO: 5)is identical with CBF1 [Stockinger, E. J. et al., Proc. Natl. Acad. Sci.USA 94:1035-1040 (1997)], but DREB1C gene (SEQ ID NO: 7) and DREB2B gene(SEQ ID NO: 9) were found to be novel.

From the analysis of the open reading frame of DREB1C gene, it was foundthat the gene product encoded by this gene is a protein consisting of216 amino acid residues with a molecular weight of about 24.3 kDa (SEQID NO: 8). Also, it was found that the gene product encoded by DREB2Bgene is a protein consisting of 330 amino acid residues with a molecularweight of about 37.1 kDa (SEQ ID NO: 10). SEQ ID NO:6 is the amino acidsequence of DREB1B protein.

EXAMPLE 3

Analysis of the DRE-Binding Ability of DREB1A and DREB2A Proteins

The ability of DREB1A and DREB2A proteins to bind to DRE was analyzed bypreparing a fusion protein composed of glutathione-S-transferase (GST)and DREB1A or DREB2A protein using E. coli and then performing a gelshift assay. Briefly, the 429 bp DNA fragment located from position 119to position 547 of the nucleotide sequence of DREB1A cDNA or the 500 bpDNA fragment located from position 167 to position 666 of the nucleotidesequence of DREB2A cDNA was amplified by PCR. Then, the amplifiedfragment was ligated to the EcoRI-SalI site of plasmid pGEX-4T-1(Pharmacia). After the introduction of this plasmid into E. coli JM109,the resultant transformant was cultured in 200 ml of 2×YT medium(Molecular Cloning, (1982) Cold Spring Harbor Laboratory Press). To thisculture, 1 mM isopropyl β-D-thiogalactoside which activates the promoterof plasmid pGEX-4T-1 was added to thereby induce the synthesis of afusion protein of DREB1A (or DREB2A) and GST.

E. coli in which the fusion protein had been induced was lysed in 13 mlof buffer (10 mM Tris-HCl, 0.1 mM DTT, 0.1 mM phenylmethylsulfonylfluoride). Then, 1% Triton X-100 and 1 mM EDTA were added thereto. Afterthe cells were disrupted by sonication, the disrupted material wascentrifuged at 22,000 g for 20 min. Then, the fusion protein waspurified with glutathione-Sepharose (Pharmacia). The resultant fusionprotein was mixed with the DRE-containing 71 bp DNA fragment labelledwith ³²P as a probe, and incubated at room temperature for 20 min. Thismixture was electrophoresed using 6% acryl amide gel containing0.25×Tris-borate-EDTA at 100 V for 2 hr. As a result of this gel shiftanalysis, those bands which migrated behind were detected. When a DNAfragment containing a varied DRE sequence was used, such bands were notdetected. Thus, it became evident that DREB1A and DREB2A proteinsspecifically bound to DRE sequence (FIG. 2).

EXAMPLE 4

Analysis of the Ability of DREB1A and DREB2A Proteins to Activate theTranscription of Genes Located Downstream of DRE

In order to examine whether DREB1A and DREB2A proteins are able totrans-activate DRE-dependent transcription in plant cells, atrans-activation experiment was conducted using a protoplast systemprepared from Arabidopsis thaliana leaves. Briefly, the cDNA of DREB1Aor DREB2A was ligated to a pBI221 plasmid containing CaMV35S promoter tothereby construct an effector plasmid.

On the other hand, 3 cassettes of the DRE-containing 71 base DNA regionwere connected tandemly to prepare a DNA fragment, which was ligatedupstream to the minimum TATA promoter located upstream ofβ-glucuronidase (GUS) gene in a plasmid derived from pBI221 plasmid toconstruct a reporter plasmid. Subsequently, these two plasmids wereintroduced into protoplasts from Arabidopsis thaliana and then GUSactivity was determined. When DREB1A or DREB2A protein was expressedsimultaneously, GUS activity increased. This shows that DREB1A andDREB2A proteins are transcription factors which activate transcriptionthrough DRE sequence (FIG. 3).

EXAMPLE 5

(1) Construction of a Plant Plasmid

Plasmid pSKDREB1A (10 μg) obtained as described above was digested withEcoRV (20 U) and SmaI (20 U) in a buffer containing 10 mM Tris-HCl (pH7.5), 10 mM MgCl₂, 1 mM dithiothreitol (DTT) and 100 mM NaCl at 37° C.for 2 hr to thereby obtain a DNA fragment of about 0.9 kb containingDREB1A gene. On the other hand, plasmid pBI2113Not (10 μg) containingpromoter DNA was digested with SmaI in a buffer containing 10 mMTris-HCl (pH 7.5), 10 MM MgCl₂, 1 mM DTT and 100 mM NaCl at 37° C. for 2hr. The 0.9 kb DNA fragment containing DREB1A gene obtained by digestionand the digested pBI2113Not were treated with T4 DNA ligase (2 U) in abuffer [66 mM Tris-HCl (pH 7.6), 6.6 mM MgCl₂, 10 mM DTT, 0.1 mM ATP] at15° C. for 16 hr. The resultant DNA was transformed into E. coli JM109,from which plasmid pBI35S:DREB1A was obtained. With respect to thedirection of DREB1A gene, those plasmids in which this gene was ligatedin the sense direction were selected by determining the nucleotidesequence at the junction site of plasmid pBI35S:DREB1A. PlasmidpBI2113Not mentioned above is a plasmid prepared by digestingpBI2113[Plant Cell Physiology 37:49-59 (1996)] with SmaI and SacI toremove the coding region of GUS gene and ligating a SmaI-NotI-Sacpolylinker to the resultant plasmid. The plant plasmid pBI35S:DREB1Aprepared as described above was transformed into E. coli DH5a (FIG. 4).

Briefly, the plant plasmid pBI35S:DREB1A, E. coli DH5α, helper plasmidpRK2013-containing E. coli HB101 and Agrobacterium C58 were mixed andcultured on LB agar medium at 28° C. for 24 hr. Grown colonies werescraped off and suspended in 1 ml of LB medium. This suspension (10 ml)was plated on LB agar medium containing 100 μg/ml rifampicin and 20μg/mlkanamycin and cultured at 28° C. for 2 days to thereby obtain a zygoteAgrobacterium C58 (pBI35S:DREB1A).

(2) Gene Transfer into Arabidopsis thaliana by Agrobacterium Infection.

The resultant Agrobacterium was cultured in 10 ml of LB mediumcontaining 100 μg/ml rifampicin and 20 μg/ml kanamycin at 28° C. for 24hr. Further, this culture fluid was added to 500 ml of LB medium andcultured for another 24 hr. The resultant culture fluid was centrifugedto remove the medium, and the cell pellet was suspended in 250 ml of LBmedium.

On the other hand, 4 to 5 Arabidopsis thaliana plant bodies were grownin 9 cm pots containing soil composed of vermiculite and perlite (50:50)for 6 weeks. Then, the plant body was directly dipped in the LB culturefluid containing the Agrobacterium bearing plasmid pBI35S:DREB1A andplaced in a desiccator, which was sucked with a vacuum pump to reducethe pressure to 650 mmHg and then left for 10 min. Subsequently, theplant pot was transferred to a tray and covered with a wrap to maintainthe humidity. The next day, the wrap was removed. Thereafter, the plantwas grown uncovered to thereby obtain seeds. After sterilization in anaqueous solution of sodium hypochlorite, the seeds were sown on an agarmedium for selection (MS medium supplemented with 100 μg/ml vancomycinand 30 μg/ml kanamycin). Arabidopsis thaliana seedlings grown on thismedium were transplanted to pots and grown there to obtain seeds of thetransformed plant.

(3) Identification of Genes Whose Expression Has Been Altered by theIntroduced Gene and the Transcription Factor Encoded by the Gene

Genes whose expression is considered to have been altered by theintroduced gene DREB1A and the transcription factor encoded by this genein the transformed plant were identified by Northern blot analysis. Inthis analysis, transcriptional activation of DREB1A, rd29A, kin1,cor6.6, cor15a, rd17, erd10, P5CS, erd1, rd22 and rd29B genes wereinvestigated. Transformed and wild type Arabidopsis thaliana plants wereused for comparing the expression of the above genes. Two grams of plantbodies grown on GM agar medium for 3 weeks were exposed to dehydrationstress and low temperature stress. Dehydration stress was given bypulling out the plant from the agar medium and drying it on a filterpaper for 5 hr. Low temperature stress was given by retaining the plantat 4° C. for 5 hr. Total RNA was prepared separately from control plantswhich was given no stress and plants which were given dehydration andlow temperature stresses. The resultant total RNA was subjected toelectrophoresis. Then, expressing genes were assayed by Northern blotanalysis. Generally, introduced genes are located on the genome of atransformed plant in a similar manner. However, due to the difference inthe locations on the genome, the expression of the introduced genesvaries. This is a phenomenon called position effect. By assayingtransformants by Northern blotting with a DNA fragment from theintroduced gene as a probe, those transformants in which the introducedgene was expressed more highly could be selected. Also, by using a DNAfragment of the gene involved in stress tolerance as a probe, stresstolerance genes which exhibit changes when DREB1A gene is introducedcould be identified (FIG. 5).

EXAMPLE 6 Expression of Dehydration/Freezing Tolerance

Dehydration/freezing tolerance was investigated on Arabidopsis thalianatransformant comprising DREB1A gene which had been grown in 9 cm potscontaining soil composed of vermiculite and perlite (50:50) for 3 weeks.As a control, Arabidopsis thaliana transformed with pBI121 notcontaining DREB1A gene was used. As to dehydration tolerance, watersupply was stopped for 2 weeks and then plant survival was examined. Asto freezing tolerance, the plant was maintained at −6° C. for 2 days andthen grown at 22° C. for 5 days. Thereafter, its survival ratio wasexamined.

As a result, all the control plants were withered but the transgenicplants into which DREB1A gene is introduced exhibited a high survivalratio (FIG. 6).

This specification includes part or all of the contents as disclosed inthe specification and/or drawings of Japanese Patent Application No.10-228457, which is a priority document of the present application.

All publications, patents and patent applications cited herein areincorporated herein by reference in their entirety.

EFFECT OF THE INVENTION

According to the present invention, a protein which binds to DRE toactivate the transcription of genes located downstream of the DRE, agene encoding the protein, a recombinant vector comprising the gene, atransformant comprising the recombinant vector, a transgenic plantcomprising the gene, and a method for producing the gene using thetransformant are provided. The present invention is useful for creatinga stress tolerant plant.

SEQUENCE LISTING <160> NUMBER OF SEQ ID NOS: 12 <210> SEQ ID NO 1 <211>LENGTH: 933 <212> TYPE: DNA <213> ORGANISM: Arabidopsis thaliana <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (119)..(766) <400>SEQUENCE: 1 cctgaactag aacagaaaga gagagaaact attatttcag caaaccataccaacaaaaaa 60 gacagagatc ttttagttac cttatccagt ttcttgaaac agagtactcttctgatca 118 atg aac tca ttt tct gct ttt tct gaa atg ttt ggc tcc gat tacgag 166 Met Asn Ser Phe Ser Ala Phe Ser Glu Met Phe Gly Ser Asp Tyr Glu1 5 10 15 tct tcg gtt tcc tca ggc ggt gat tat att ccg acg ctt gcg agcagc 214 Ser Ser Val Ser Ser Gly Gly Asp Tyr Ile Pro Thr Leu Ala Ser Ser20 25 30 tgc ccc aag aaa ccg gcg ggt cgt aag aag ttt cgt gag act cgt cac262 Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg Glu Thr Arg His 3540 45 cca ata tac aga gga gtt cgt cgg aga aac tcc ggt aag tgg gtt tgt310 Pro Ile Tyr Arg Gly Val Arg Arg Arg Asn Ser Gly Lys Trp Val Cys 5055 60 gag gtt aga gaa cca aac aag aaa aca agg att tgg ctc gga aca ttt358 Glu Val Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe 6570 75 80 caa acc gct gag atg gca gct cga gct cac gac gtt gcc gct tta gcc406 Gln Thr Ala Glu Met Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala 8590 95 ctt cgt ggc cga tca gcc tgt ctc aat ttc gct gac tcg gct tgg aga454 Leu Arg Gly Arg Ser Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg 100105 110 ctc cga atc ccg gaa tca act tgc gct aag gac atc caa aag gcg gcg502 Leu Arg Ile Pro Glu Ser Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala 115120 125 gct gaa gct gcg ttg gcg ttt cag gat gag atg tgt gat gcg acg acg550 Ala Glu Ala Ala Leu Ala Phe Gln Asp Glu Met Cys Asp Ala Thr Thr 130135 140 gat cat ggc ttc gac atg gag gag acg ttg gtg gag gct att tac acg598 Asp His Gly Phe Asp Met Glu Glu Thr Leu Val Glu Ala Ile Tyr Thr 145150 155 160 gcg gaa cag agc gaa aat gcg ttt tat atg cac gat gag gcg atgttt 646 Ala Glu Gln Ser Glu Asn Ala Phe Tyr Met His Asp Glu Ala Met Phe165 170 175 gag atg ccg agt ttg ttg gct aat atg gca gaa ggg atg ctt ttgccg 694 Glu Met Pro Ser Leu Leu Ala Asn Met Ala Glu Gly Met Leu Leu Pro180 185 190 ctt ccg tcc gta cag tgg aat cat aat cat gaa gtc gac ggc gatgat 742 Leu Pro Ser Val Gln Trp Asn His Asn His Glu Val Asp Gly Asp Asp195 200 205 gac gac gta tcg tta tgg agt tat taaaactcag attattatttccatttttag 796 Asp Asp Val Ser Leu Trp Ser Tyr 210 215 tacgatactttttattttat tattattttt agatcctttt ttagaatgga atcttcatta 856 tgtttgtaaaactgagaaac gagtgtaaat taaattgatt cagtttcagt ataaaaaaaa 916 aaaaaaaaaaaaaaaaa 933 <210> SEQ ID NO 2 <211> LENGTH: 216 <212> TYPE: PRT <213>ORGANISM: Arabidopsis thaliana <400> SEQUENCE: 2 Met Asn Ser Phe Ser AlaPhe Ser Glu Met Phe Gly Ser Asp Tyr Glu 1 5 10 15 Ser Ser Val Ser SerGly Gly Asp Tyr Ile Pro Thr Leu Ala Ser Ser 20 25 30 Cys Pro Lys Lys ProAla Gly Arg Lys Lys Phe Arg Glu Thr Arg His 35 40 45 Pro Ile Tyr Arg GlyVal Arg Arg Arg Asn Ser Gly Lys Trp Val Cys 50 55 60 Glu Val Arg Glu ProAsn Lys Lys Thr Arg Ile Trp Leu Gly Thr Phe 65 70 75 80 Gln Thr Ala GluMet Ala Ala Arg Ala His Asp Val Ala Ala Leu Ala 85 90 95 Leu Arg Gly ArgSer Ala Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg 100 105 110 Leu Arg IlePro Glu Ser Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala 115 120 125 Ala GluAla Ala Leu Ala Phe Gln Asp Glu Met Cys Asp Ala Thr Thr 130 135 140 AspHis Gly Phe Asp Met Glu Glu Thr Leu Val Glu Ala Ile Tyr Thr 145 150 155160 Ala Glu Gln Ser Glu Asn Ala Phe Tyr Met His Asp Glu Ala Met Phe 165170 175 Glu Met Pro Ser Leu Leu Ala Asn Met Ala Glu Gly Met Leu Leu Pro180 185 190 Leu Pro Ser Val Gln Trp Asn His Asn His Glu Val Asp Gly AspAsp 195 200 205 Asp Asp Val Ser Leu Trp Ser Tyr 210 215 <210> SEQ ID NO3 <211> LENGTH: 1437 <212> TYPE: DNA <213> ORGANISM: Arabidopsisthaliana <220> FEATURE: <221> NAME/KEY: CDS <222> LOCATION:(167)..(1171) <400> SEQUENCE: 3 gctgtctgat aaaaagaaga ggaaaactcgaaaaagctac acacaagaag aagaagaaaa 60 gatacgagca agaagactaa acacgaaagcgatttatcaa ctcgaaggaa gagactttga 120 ttttcaaatt tcgtccccta tagattgtgttgtttctggg aaggag atg gca gtt 175 Met Ala Val 1 tat gat cag agt gga gataga aac aga aca caa att gat aca tcg agg 223 Tyr Asp Gln Ser Gly Asp ArgAsn Arg Thr Gln Ile Asp Thr Ser Arg 5 10 15 aaa agg aaa tct aga agt agaggt gac ggt act act gtg gct gag aga 271 Lys Arg Lys Ser Arg Ser Arg GlyAsp Gly Thr Thr Val Ala Glu Arg 20 25 30 35 tta aag aga tgg aaa gag tataac gag acc gta gaa gaa gtt tct acc 319 Leu Lys Arg Trp Lys Glu Tyr AsnGlu Thr Val Glu Glu Val Ser Thr 40 45 50 aag aag agg aaa gta cct gcg aaaggg tcg aag aag ggt tgt atg aaa 367 Lys Lys Arg Lys Val Pro Ala Lys GlySer Lys Lys Gly Cys Met Lys 55 60 65 ggt aaa gga gga cca gag aat agc cgatgt agt ttc aga gga gtt agg 415 Gly Lys Gly Gly Pro Glu Asn Ser Arg CysSer Phe Arg Gly Val Arg 70 75 80 caa agg att tgg ggt aaa tgg gtt gct gagatc aga gag cct aat cga 463 Gln Arg Ile Trp Gly Lys Trp Val Ala Glu IleArg Glu Pro Asn Arg 85 90 95 ggt agc agg ctt tgg ctt ggt act ttc cct actgct caa gaa gct gct 511 Gly Ser Arg Leu Trp Leu Gly Thr Phe Pro Thr AlaGln Glu Ala Ala 100 105 110 115 tct gct tat gat gag gct gct aaa gct atgtat ggt cct ttg gct cgt 559 Ser Ala Tyr Asp Glu Ala Ala Lys Ala Met TyrGly Pro Leu Ala Arg 120 125 130 ctt aat ttc cct cgg tct gat gcg tct gaggtt acg agt acc tca agt 607 Leu Asn Phe Pro Arg Ser Asp Ala Ser Glu ValThr Ser Thr Ser Ser 135 140 145 cag tct gag gtg tgt act gtt gag act cctggt tgt gtt cat gtg aaa 655 Gln Ser Glu Val Cys Thr Val Glu Thr Pro GlyCys Val His Val Lys 150 155 160 aca gag gat cca gat tgt gaa tct aaa cccttc tcc ggt gga gtg gag 703 Thr Glu Asp Pro Asp Cys Glu Ser Lys Pro PheSer Gly Gly Val Glu 165 170 175 ccg atg tat tgt ctg gag aat ggt gcg gaagag atg aag aga ggt gtt 751 Pro Met Tyr Cys Leu Glu Asn Gly Ala Glu GluMet Lys Arg Gly Val 180 185 190 195 aaa gcg gat aag cat tgg ctg agc gagttt gaa cat aac tat tgg agt 799 Lys Ala Asp Lys His Trp Leu Ser Glu PheGlu His Asn Tyr Trp Ser 200 205 210 gat att ctg aaa gag aaa gag aaa cagaag gag caa ggg att gta gaa 847 Asp Ile Leu Lys Glu Lys Glu Lys Gln LysGlu Gln Gly Ile Val Glu 215 220 225 acc tgt cag caa caa cag cag gat tcgcta tct gtt gca gac tat ggt 895 Thr Cys Gln Gln Gln Gln Gln Asp Ser LeuSer Val Ala Asp Tyr Gly 230 235 240 tgg ccc aat gat gtg gat cag agt cacttg gat tct tca gac atg ttt 943 Trp Pro Asn Asp Val Asp Gln Ser His LeuAsp Ser Ser Asp Met Phe 245 250 255 gat gtc gat gag ctt cta cgt gac ctaaat ggc gac gat gtg ttt gca 991 Asp Val Asp Glu Leu Leu Arg Asp Leu AsnGly Asp Asp Val Phe Ala 260 265 270 275 ggc tta aat cag gac cgg tac ccgggg aac agt gtt gcc aac ggt tca 1039 Gly Leu Asn Gln Asp Arg Tyr Pro GlyAsn Ser Val Ala Asn Gly Ser 280 285 290 tac agg ccc gag agt caa caa agtggt ttt gat ccg cta caa agc ctc 1087 Tyr Arg Pro Glu Ser Gln Gln Ser GlyPhe Asp Pro Leu Gln Ser Leu 295 300 305 aac tac gga ata cct ccg ttt cagctc gag gga aag gat ggt aat gga 1135 Asn Tyr Gly Ile Pro Pro Phe Gln LeuGlu Gly Lys Asp Gly Asn Gly 310 315 320 ttc ttc gac gac ttg agt tac ttggat ctg gag aac taaacaaaac 1181 Phe Phe Asp Asp Leu Ser Tyr Leu Asp LeuGlu Asn 325 330 335 aatatgaagc tttttggatt tgatatttgc cttaatcccacaacgactgt tgattctcta 1241 tccgagtttt agtgatatag agaactacag aacacgttttttcttgttat aaaggtgaac 1301 tgtatatatc gaaacagtga tatgacaata gagaagacaactatagtttg ttagtctgct 1361 tctcttaagt tgttctttag atatgtttta tgttttgtaacaacaggaat gaataataca 1421 cacttgtaaa aaaaaa 1437 <210> SEQ ID NO 4<211> LENGTH: 335 <212> TYPE: PRT <213> ORGANISM: Arabidopsis thaliana<400> SEQUENCE: 4 Met Ala Val Tyr Asp Gln Ser Gly Asp Arg Asn Arg ThrGln Ile Asp 1 5 10 15 Thr Ser Arg Lys Arg Lys Ser Arg Ser Arg Gly AspGly Thr Thr Val 20 25 30 Ala Glu Arg Leu Lys Arg Trp Lys Glu Tyr Asn GluThr Val Glu Glu 35 40 45 Val Ser Thr Lys Lys Arg Lys Val Pro Ala Lys GlySer Lys Lys Gly 50 55 60 Cys Met Lys Gly Lys Gly Gly Pro Glu Asn Ser ArgCys Ser Phe Arg 65 70 75 80 Gly Val Arg Gln Arg Ile Trp Gly Lys Trp ValAla Glu Ile Arg Glu 85 90 95 Pro Asn Arg Gly Ser Arg Leu Trp Leu Gly ThrPhe Pro Thr Ala Gln 100 105 110 Glu Ala Ala Ser Ala Tyr Asp Glu Ala AlaLys Ala Met Tyr Gly Pro 115 120 125 Leu Ala Arg Leu Asn Phe Pro Arg SerAsp Ala Ser Glu Val Thr Ser 130 135 140 Thr Ser Ser Gln Ser Glu Val CysThr Val Glu Thr Pro Gly Cys Val 145 150 155 160 His Val Lys Thr Glu AspPro Asp Cys Glu Ser Lys Pro Phe Ser Gly 165 170 175 Gly Val Glu Pro MetTyr Cys Leu Glu Asn Gly Ala Glu Glu Met Lys 180 185 190 Arg Gly Val LysAla Asp Lys His Trp Leu Ser Glu Phe Glu His Asn 195 200 205 Tyr Trp SerAsp Ile Leu Lys Glu Lys Glu Lys Gln Lys Glu Gln Gly 210 215 220 Ile ValGlu Thr Cys Gln Gln Gln Gln Gln Asp Ser Leu Ser Val Ala 225 230 235 240Asp Tyr Gly Trp Pro Asn Asp Val Asp Gln Ser His Leu Asp Ser Ser 245 250255 Asp Met Phe Asp Val Asp Glu Leu Leu Arg Asp Leu Asn Gly Asp Asp 260265 270 Val Phe Ala Gly Leu Asn Gln Asp Arg Tyr Pro Gly Asn Ser Val Ala275 280 285 Asn Gly Ser Tyr Arg Pro Glu Ser Gln Gln Ser Gly Phe Asp ProLeu 290 295 300 Gln Ser Leu Asn Tyr Gly Ile Pro Pro Phe Gln Leu Glu GlyLys Asp 305 310 315 320 Gly Asn Gly Phe Phe Asp Asp Leu Ser Tyr Leu AspLeu Glu Asn 325 330 335 <210> SEQ ID NO 5 <211> LENGTH: 937 <212> TYPE:DNA <213> ORGANISM: Arabidopsis thaliana <220> FEATURE: <221> NAME/KEY:CDS <222> LOCATION: (164)..(802) <400> SEQUENCE: 5 cttgaaaaag aatctacctgaaaagaaaaa aaagagagag agatataaat agctttacca 60 agacagatat actatcttttattaatccaa aaagactgag aactctagta actacgtact 120 acttaaacct tatccagtttcttgaaacag agtactctga tca atg aac tca ttt 175 Met Asn Ser Phe 1 tca gctttt tct gaa atg ttt ggc tcc gat tac gag cct caa ggc gga 223 Ser Ala PheSer Glu Met Phe Gly Ser Asp Tyr Glu Pro Gln Gly Gly 5 10 15 20 gat tattgt ccg acg ttg gcc acg agt tgt ccg aag aaa ccg gcg ggc 271 Asp Tyr CysPro Thr Leu Ala Thr Ser Cys Pro Lys Lys Pro Ala Gly 25 30 35 cgt aag aagttt cgt gag act cgt cac cca att tac aga gga gtt cgt 319 Arg Lys Lys PheArg Glu Thr Arg His Pro Ile Tyr Arg Gly Val Arg 40 45 50 caa aga aac tccggt aag tgg gtt tct gaa gtg aga gag cca aac aag 367 Gln Arg Asn Ser GlyLys Trp Val Ser Glu Val Arg Glu Pro Asn Lys 55 60 65 aaa acc agg att tggctc ggg act ttc caa acc gct gag atg gca gct 415 Lys Thr Arg Ile Trp LeuGly Thr Phe Gln Thr Ala Glu Met Ala Ala 70 75 80 cgt gct cac gac gtc gctgca tta gcc ctc cgt ggc cga tca gca tgt 463 Arg Ala His Asp Val Ala AlaLeu Ala Leu Arg Gly Arg Ser Ala Cys 85 90 95 100 ctc aac ttc gct gac tcggct tgg cgg cta cga atc ccg gag tca aca 511 Leu Asn Phe Ala Asp Ser AlaTrp Arg Leu Arg Ile Pro Glu Ser Thr 105 110 115 tgc gcc aag gat atc caaaaa gcg gct gct gaa gcg gcg ttg gct ttt 559 Cys Ala Lys Asp Ile Gln LysAla Ala Ala Glu Ala Ala Leu Ala Phe 120 125 130 caa gat gag acg tgt gatacg acg acc acg aat cat ggc ctg gac atg 607 Gln Asp Glu Thr Cys Asp ThrThr Thr Thr Asn His Gly Leu Asp Met 135 140 145 gag gag acg atg gtg gaagct att tat aca ccg gaa cag agc gaa ggt 655 Glu Glu Thr Met Val Glu AlaIle Tyr Thr Pro Glu Gln Ser Glu Gly 150 155 160 gcg ttt tat atg gat gaggag aca atg ttt ggg atg ccg act ttg ttg 703 Ala Phe Tyr Met Asp Glu GluThr Met Phe Gly Met Pro Thr Leu Leu 165 170 175 180 gat aat atg gct gaaggc atg ctt tta ccg ccg ccg tct gtt caa tgg 751 Asp Asn Met Ala Glu GlyMet Leu Leu Pro Pro Pro Ser Val Gln Trp 185 190 195 aat cat aat tat gacggc gaa gga gat ggt gac gtg tcg ctt tgg agt 799 Asn His Asn Tyr Asp GlyGlu Gly Asp Gly Asp Val Ser Leu Trp Ser 200 205 210 tac taatattcgatagtcgtttc catttttgta ctatagtttg aaaatattct 852 Tyr agttcctttttttagaatgg ttccttcatt ttattttatt ttattgttgt agaaacgagt 912 ggaaaataattcaatacaaa aaaaa 937 <210> SEQ ID NO 6 <211> LENGTH: 213 <212> TYPE: PRT<213> ORGANISM: Arabidopsis thaliana <400> SEQUENCE: 6 Met Asn Ser PheSer Ala Phe Ser Glu Met Phe Gly Ser Asp Tyr Glu 1 5 10 15 Pro Gln GlyGly Asp Tyr Cys Pro Thr Leu Ala Thr Ser Cys Pro Lys 20 25 30 Lys Pro AlaGly Arg Lys Lys Phe Arg Glu Thr Arg His Pro Ile Tyr 35 40 45 Arg Gly ValArg Gln Arg Asn Ser Gly Lys Trp Val Ser Glu Val Arg 50 55 60 Glu Pro AsnLys Lys Thr Arg Ile Trp Leu Gly Thr Phe Gln Thr Ala 65 70 75 80 Glu MetAla Ala Arg Ala His Asp Val Ala Ala Leu Ala Leu Arg Gly 85 90 95 Arg SerAla Cys Leu Asn Phe Ala Asp Ser Ala Trp Arg Leu Arg Ile 100 105 110 ProGlu Ser Thr Cys Ala Lys Asp Ile Gln Lys Ala Ala Ala Glu Ala 115 120 125Ala Leu Ala Phe Gln Asp Glu Thr Cys Asp Thr Thr Thr Thr Asn His 130 135140 Gly Leu Asp Met Glu Glu Thr Met Val Glu Ala Ile Tyr Thr Pro Glu 145150 155 160 Gln Ser Glu Gly Ala Phe Tyr Met Asp Glu Glu Thr Met Phe GlyMet 165 170 175 Pro Thr Leu Leu Asp Asn Met Ala Glu Gly Met Leu Leu ProPro Pro 180 185 190 Ser Val Gln Trp Asn His Asn Tyr Asp Gly Glu Gly AspGly Asp Val 195 200 205 Ser Leu Trp Ser Tyr 210 <210> SEQ ID NO 7 <211>LENGTH: 944 <212> TYPE: DNA <213> ORGANISM: Arabidopsis thaliana <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (135)..(782) <400>SEQUENCE: 7 cctgaattag aaaagaaaga tagatagaga aataaatatt ttatcataccatacaaaaaa 60 agacagagat cttctactta ctctactctc ataaacctta tccagtttcttgaaacagag 120 tactcttctg atca atg aac tca ttt tct gcc ttt tct gaa atgttt ggc 170 Met Asn Ser Phe Ser Ala Phe Ser Glu Met Phe Gly 1 5 10 tccgat tac gag tct ccg gtt tcc tca ggc ggt gat tac agt ccg aag 218 Ser AspTyr Glu Ser Pro Val Ser Ser Gly Gly Asp Tyr Ser Pro Lys 15 20 25 ctt gccacg agc tgc ccc aag aaa cca gcg gga agg aag aag ttt cgt 266 Leu Ala ThrSer Cys Pro Lys Lys Pro Ala Gly Arg Lys Lys Phe Arg 30 35 40 gag act cgtcac cca att tac aga gga gtt cgt caa aga aac tcc ggt 314 Glu Thr Arg HisPro Ile Tyr Arg Gly Val Arg Gln Arg Asn Ser Gly 45 50 55 60 aag tgg gtgtgt gag ttg aga gag cca aac aag aaa acg agg att tgg 362 Lys Trp Val CysGlu Leu Arg Glu Pro Asn Lys Lys Thr Arg Ile Trp 65 70 75 ctc ggg act ttccaa acc gct gag atg gca gct cgt gct cac gac gtc 410 Leu Gly Thr Phe GlnThr Ala Glu Met Ala Ala Arg Ala His Asp Val 80 85 90 gcc gcc ata gct ctccgt ggc aga tct gcc tgt ctc aat ttc gct gac 458 Ala Ala Ile Ala Leu ArgGly Arg Ser Ala Cys Leu Asn Phe Ala Asp 95 100 105 tcg gct tgg cgg ctacga atc ccg gaa tca acc tgt gcc aag gaa atc 506 Ser Ala Trp Arg Leu ArgIle Pro Glu Ser Thr Cys Ala Lys Glu Ile 110 115 120 caa aag gcg gcg gctgaa gcc gcg ttg aat ttt caa gat gag atg tgt 554 Gln Lys Ala Ala Ala GluAla Ala Leu Asn Phe Gln Asp Glu Met Cys 125 130 135 140 cat atg acg acggat gct cat ggt ctt gac atg gag gag acc ttg gtg 602 His Met Thr Thr AspAla His Gly Leu Asp Met Glu Glu Thr Leu Val 145 150 155 gag gct att tatacg ccg gaa cag agc caa gat gcg ttt tat atg gat 650 Glu Ala Ile Tyr ThrPro Glu Gln Ser Gln Asp Ala Phe Tyr Met Asp 160 165 170 gaa gag gcg atgttg ggg atg tct agt ttg ttg gat aac atg gcc gaa 698 Glu Glu Ala Met LeuGly Met Ser Ser Leu Leu Asp Asn Met Ala Glu 175 180 185 ggg atg ctt ttaccg tcg ccg tcg gtt caa tgg aac tat aat ttt gat 746 Gly Met Leu Leu ProSer Pro Ser Val Gln Trp Asn Tyr Asn Phe Asp 190 195 200 gtc gag gga gatgat gac gtg tcc tta tgg agc tat taaaattcga 792 Val Glu Gly Asp Asp AspVal Ser Leu Trp Ser Tyr 205 210 215 tttttatttc catttttggt attatagctttttatacatt tgatcctttt ttagaatgga 852 tcttcttctt tttttggttg tgagaaacgaatgtaaatgg taaaagttgt tgtcaaatgc 912 aaatgttttt gagtgcagaa tatataatct tt944 <210> SEQ ID NO 8 <211> LENGTH: 216 <212> TYPE: PRT <213> ORGANISM:Arabidopsis thaliana <400> SEQUENCE: 8 Met Asn Ser Phe Ser Ala Phe SerGlu Met Phe Gly Ser Asp Tyr Glu 1 5 10 15 Ser Pro Val Ser Ser Gly GlyAsp Tyr Ser Pro Lys Leu Ala Thr Ser 20 25 30 Cys Pro Lys Lys Pro Ala GlyArg Lys Lys Phe Arg Glu Thr Arg His 35 40 45 Pro Ile Tyr Arg Gly Val ArgGln Arg Asn Ser Gly Lys Trp Val Cys 50 55 60 Glu Leu Arg Glu Pro Asn LysLys Thr Arg Ile Trp Leu Gly Thr Phe 65 70 75 80 Gln Thr Ala Glu Met AlaAla Arg Ala His Asp Val Ala Ala Ile Ala 85 90 95 Leu Arg Gly Arg Ser AlaCys Leu Asn Phe Ala Asp Ser Ala Trp Arg 100 105 110 Leu Arg Ile Pro GluSer Thr Cys Ala Lys Glu Ile Gln Lys Ala Ala 115 120 125 Ala Glu Ala AlaLeu Asn Phe Gln Asp Glu Met Cys His Met Thr Thr 130 135 140 Asp Ala HisGly Leu Asp Met Glu Glu Thr Leu Val Glu Ala Ile Tyr 145 150 155 160 ThrPro Glu Gln Ser Gln Asp Ala Phe Tyr Met Asp Glu Glu Ala Met 165 170 175Leu Gly Met Ser Ser Leu Leu Asp Asn Met Ala Glu Gly Met Leu Leu 180 185190 Pro Ser Pro Ser Val Gln Trp Asn Tyr Asn Phe Asp Val Glu Gly Asp 195200 205 Asp Asp Val Ser Leu Trp Ser Tyr 210 215 <210> SEQ ID NO 9 <211>LENGTH: 1513 <212> TYPE: DNA <213> ORGANISM: Arabidopsis thaliana <220>FEATURE: <221> NAME/KEY: CDS <222> LOCATION: (183)..(1172) <221>NAME/KEY: modified_base <222> LOCATION: 1440 <223> OTHER INFORMATION: nrepresents a, g, c or t <221> NAME/KEY: modified_base <222> LOCATION:1443 <223> OTHER INFORMATION: n represents a, g, c or t <221> NAME/KEY:modified_base <222> LOCATION: 1444 <223> OTHER INFORMATION: n representsa, g, c or t <221> NAME/KEY: modified_base <222> LOCATION: 1447 <223>OTHER INFORMATION: n represents a, g, c or t <221> NAME/KEY:modified_base <222> LOCATION: 1450 <223> OTHER INFORMATION: n representsa, g, c or t <221> NAME/KEY: modified_base <222> LOCATION: 1459 <223>OTHER INFORMATION: n represents a, g, c or t <221> NAME/KEY:modified_base <222> LOCATION: 1472 <223> OTHER INFORMATION: n representsa, g, c or t <221> NAME/KEY: modified_base <222> LOCATION: 1495 <223>OTHER INFORMATION: n represents a, g, c or t <221> NAME/KEY:modified_base <222> LOCATION: 1508 <223> OTHER INFORMATION: n representsa, g, c or t <221> NAME/KEY: modified_base <222> LOCATION: 1510 <223>OTHER INFORMATION: n represents a, g, c or t <400> SEQUENCE: 9gagacgctag aaagaacgcg aaagcttgcg aagaagattt gcttttgatc gacttaacac 60gaacaacaaa caacatctgc gtgataaaga agagattttt gcctaaataa agaagagatt 120cgactctaat cctggagtta tcattcacga tagattctta gattgcgact ataaagaaga 180 agatg gct gta tat gaa caa acc gga acc gag cag ccg aag aaa agg 227 Met AlaVal Tyr Glu Gln Thr Gly Thr Glu Gln Pro Lys Lys Arg 1 5 10 15 aaa tctagg gct cga gca ggt ggt tta acg gtg gct gat agg cta aag 275 Lys Ser ArgAla Arg Ala Gly Gly Leu Thr Val Ala Asp Arg Leu Lys 20 25 30 aag tgg aaagag tac aac gag att gtt gaa gct tcg gct gtt aaa gaa 323 Lys Trp Lys GluTyr Asn Glu Ile Val Glu Ala Ser Ala Val Lys Glu 35 40 45 gga gag aaa ccgaaa cgc aaa gtt cct gcg aaa ggg tcg aag aaa ggt 371 Gly Glu Lys Pro LysArg Lys Val Pro Ala Lys Gly Ser Lys Lys Gly 50 55 60 tgt atg aag ggt aaagga gga cca gat aat tct cac tgt agt ttt aga 419 Cys Met Lys Gly Lys GlyGly Pro Asp Asn Ser His Cys Ser Phe Arg 65 70 75 gga gtt aga caa agg atttgg ggt aaa tgg gtt gca gag att cga gaa 467 Gly Val Arg Gln Arg Ile TrpGly Lys Trp Val Ala Glu Ile Arg Glu 80 85 90 95 ccg aaa ata gga act agactt tgg ctt ggt act ttt cct acc gcg gaa 515 Pro Lys Ile Gly Thr Arg LeuTrp Leu Gly Thr Phe Pro Thr Ala Glu 100 105 110 aaa gct gct tcc gct tatgat gaa gcg gct acc gct atg tac ggt tca 563 Lys Ala Ala Ser Ala Tyr AspGlu Ala Ala Thr Ala Met Tyr Gly Ser 115 120 125 ttg gct cgt ctt aac ttccct cag tct gtt ggg tct gag ttt act agt 611 Leu Ala Arg Leu Asn Phe ProGln Ser Val Gly Ser Glu Phe Thr Ser 130 135 140 acg tct agt caa tct gaggtg tgt acg gtt gaa aat aag gcg gtt gtt 659 Thr Ser Ser Gln Ser Glu ValCys Thr Val Glu Asn Lys Ala Val Val 145 150 155 tgt ggt gat gtt tgt gtgaag cat gaa gat act gat tgt gaa tct aat 707 Cys Gly Asp Val Cys Val LysHis Glu Asp Thr Asp Cys Glu Ser Asn 160 165 170 175 cca ttt agt cag atttta gat gtt aga gaa gag tct tgt gga acc agg 755 Pro Phe Ser Gln Ile LeuAsp Val Arg Glu Glu Ser Cys Gly Thr Arg 180 185 190 ccg gac agt tgc acggtt gga cat caa gat atg aat tct tcg ctg aat 803 Pro Asp Ser Cys Thr ValGly His Gln Asp Met Asn Ser Ser Leu Asn 195 200 205 tac gat ttg ctg ttagag ttt gag cag cag tat tgg ggc caa gtt ttg 851 Tyr Asp Leu Leu Leu GluPhe Glu Gln Gln Tyr Trp Gly Gln Val Leu 210 215 220 cag gag aaa gag aaaccg aag cag gaa gaa gag gag ata cag caa cag 899 Gln Glu Lys Glu Lys ProLys Gln Glu Glu Glu Glu Ile Gln Gln Gln 225 230 235 caa cag gaa cag caacag caa cag ctg caa ccg gat ttg ctt act gtt 947 Gln Gln Glu Gln Gln GlnGln Gln Leu Gln Pro Asp Leu Leu Thr Val 240 245 250 255 gca gat tac ggttgg cct tgg tct aat gat att gta aat gat cag act 995 Ala Asp Tyr Gly TrpPro Trp Ser Asn Asp Ile Val Asn Asp Gln Thr 260 265 270 tct tgg gat cctaat gag tgc ttt gat att aat gaa ctc ctt gga gat 1043 Ser Trp Asp Pro AsnGlu Cys Phe Asp Ile Asn Glu Leu Leu Gly Asp 275 280 285 ttg aat gaa cctggt ccc cat cag agc caa gac caa aac cac gta aat 1091 Leu Asn Glu Pro GlyPro His Gln Ser Gln Asp Gln Asn His Val Asn 290 295 300 tct ggt agt tatgat ttg cat ccg ctt cat ctc gag cca cac gat ggt 1139 Ser Gly Ser Tyr AspLeu His Pro Leu His Leu Glu Pro His Asp Gly 305 310 315 cac gag ttc aatggt ttg agt tct ctg gat att tgagagttct gaggcaatgg 1192 His Glu Phe AsnGly Leu Ser Ser Leu Asp Ile 320 325 330 tcctacaaga ctacaacata atctttggattgatcatagg agaaacaaga aataggtgtt 1252 aatgatctga ttcacaatga aaaaatatttaataactcta tagtttttgt tctttccttg 1312 gatcatgaac tgttgcttct catctattgagttaatatag cgaatagcag agtttctctc 1372 tttcttctct ttgtagaaaa aaaaaaaaaaaaaaaaaaaa aaaaaaaayh sakmabgcar 1432 srcsdvsnaa nntrnatnar sarchcntrragrctrascn csrcaswash tskbabarak 1492 aantamaysa kmasrngnga c 1513 <210>SEQ ID NO 10 <211> LENGTH: 330 <212> TYPE: PRT <213> ORGANISM:Arabidopsis thaliana <400> SEQUENCE: 10 Met Ala Val Tyr Glu Gln Thr GlyThr Glu Gln Pro Lys Lys Arg Lys 1 5 10 15 Ser Arg Ala Arg Ala Gly GlyLeu Thr Val Ala Asp Arg Leu Lys Lys 20 25 30 Trp Lys Glu Tyr Asn Glu IleVal Glu Ala Ser Ala Val Lys Glu Gly 35 40 45 Glu Lys Pro Lys Arg Lys ValPro Ala Lys Gly Ser Lys Lys Gly Cys 50 55 60 Met Lys Gly Lys Gly Gly ProAsp Asn Ser His Cys Ser Phe Arg Gly 65 70 75 80 Val Arg Gln Arg Ile TrpGly Lys Trp Val Ala Glu Ile Arg Glu Pro 85 90 95 Lys Ile Gly Thr Arg LeuTrp Leu Gly Thr Phe Pro Thr Ala Glu Lys 100 105 110 Ala Ala Ser Ala TyrAsp Glu Ala Ala Thr Ala Met Tyr Gly Ser Leu 115 120 125 Ala Arg Leu AsnPhe Pro Gln Ser Val Gly Ser Glu Phe Thr Ser Thr 130 135 140 Ser Ser GlnSer Glu Val Cys Thr Val Glu Asn Lys Ala Val Val Cys 145 150 155 160 GlyAsp Val Cys Val Lys His Glu Asp Thr Asp Cys Glu Ser Asn Pro 165 170 175Phe Ser Gln Ile Leu Asp Val Arg Glu Glu Ser Cys Gly Thr Arg Pro 180 185190 Asp Ser Cys Thr Val Gly His Gln Asp Met Asn Ser Ser Leu Asn Tyr 195200 205 Asp Leu Leu Leu Glu Phe Glu Gln Gln Tyr Trp Gly Gln Val Leu Gln210 215 220 Glu Lys Glu Lys Pro Lys Gln Glu Glu Glu Glu Ile Gln Gln GlnGln 225 230 235 240 Gln Glu Gln Gln Gln Gln Gln Leu Gln Pro Asp Leu LeuThr Val Ala 245 250 255 Asp Tyr Gly Trp Pro Trp Ser Asn Asp Ile Val AsnAsp Gln Thr Ser 260 265 270 Trp Asp Pro Asn Glu Cys Phe Asp Ile Asn GluLeu Leu Gly Asp Leu 275 280 285 Asn Glu Pro Gly Pro His Gln Ser Gln AspGln Asn His Val Asn Ser 290 295 300 Gly Ser Tyr Asp Leu His Pro Leu HisLeu Glu Pro His Asp Gly His 305 310 315 320 Glu Phe Asn Gly Leu Ser SerLeu Asp Ile 325 330 <210> SEQ ID NO 11 <211> LENGTH: 30 <212> TYPE: DNA<213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Designed oligonucleotide based on the promoter region ofrd29A gene and having a HindIII site. <400> SEQUENCE: 11 aagcttaagcttacatcagt ttgaaagaaa 30 <210> SEQ ID NO 12 <211> LENGTH: 31 <212> TYPE:DNA <213> ORGANISM: Artificial Sequence <220> FEATURE: <223> OTHERINFORMATION: Designed oligonucleotide based on the promoter region ofrd29A gene and having a HindIII site. <400> SEQUENCE: 12 aagcttaagcttgctttttg gaactcatgt c 31

What is claimed is:
 1. An isolated transcription factor gene coding fora protein consisting of the amino acid sequence as shown in SEQ ID NO:2.2. An isolated gene comprising a DNA consisting of the nucleotidesequence as shown in SEQ ID NO:
 1. 3. The gene according to claim 1,wherein the protein regulates the transcription of genes locateddownstream of a stress responsive element, wherein said stress isdehydration stress, low temperature stress or salt stress.
 4. Arecombinant vector comprising the gene according to claim
 1. 5. A hostcell transformant transformed with the recombinant vector according toclaim
 4. 6. A transgenic plant transformed with the gene according toclaim 1.